JP2023515692A - Compositions and methods for the treatment of metabolic liver injury - Google Patents

Abstract

The present disclosure relates to compositions and methods for the treatment of metabolic liver injury. The compositions and methods can include an adeno-associated virus (AAV) piggyBac polynucleotide containing a transgene. The transgene can include ornithine transcarbamylase (OTC) or methylmalonyl CoA mutase (MUT1).

Description

CROSS REFERENCES TO RELATED APPLICATIONS This application is based on U.S. Provisional Application No. 62/985,047, filed March 4, 2020, and U.S. Provisional Application No. 63/121,488, filed December 4, 2020. claim priority and interest in The contents of each of the aforementioned patent applications are hereby incorporated by reference in their entirety.

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. This ASCII copy, created on March 3, 2021, is named "POTH-058_001WO_SeqList.txt" and is approximately 329KB in size.

Inherited metabolic disorders, also known as inborn errors of metabolism, are medical conditions caused by genetic defects that are most commonly inherited from parents. Normal metabolism requires a complex series of chemical reactions that cells and organisms use to convert food and other nutrients into essential compounds and energy. These chemical reactions are also used to break down and remove unwanted substances, including toxic substances. Genetic defects that cause inherited metabolic disorders often result in deficiencies in the activity of specific enzymes within one or more metabolic pathways. This deficiency can limit the subject's ability to synthesize essential compounds as well as lead to accumulation of potentially toxic substances. There are hundreds of inherited metabolic disorders that have been characterized, including those that primarily affect the liver. Inherited metabolic disorders of the liver include urea cycle disorders (UCD) and methylmalonic acidemia (MMA).

Urea cycle disorders (UCDs) result from genetic mutations that lead to defects in the metabolism of nitrogen produced by the breakdown of proteins and other nitrogen-containing molecules. UCD is commonly associated with the first four enzymes of the urea cycle: carbamoyl phosphate synthase I (CPSI), ornithine transcarbamylase (OTC), argininosuccinate synthase deficiency (ASS), and argininosuccinate lyase deficiency. (ASL), or severe or complete lack of activity of the cofactor product N-acetylglutamate synthase (NAGS), causing the accumulation of ammonia and other precursor metabolites. UCD is usually diagnosed in neonates, but late-onset UCD has been reported. UCD can lead to brain damage, cognitive impairment, and even death. Indeed, it has been hypothesized that up to 20% of sudden infant death syndrome (SIDS) cases may result from inherited metabolic disorders such as UCD.

Current treatments for UCD focus on acute control of hyperammonemia, a common symptom of UCD. Hyperammonemia is highly neurotoxic and requires intensive therapeutic intervention, including veno-venous hemofiltration. Long-term treatment of UCD currently relies on alternative pathway therapy, strict dietary protein restriction, supplementation of urea cycle intermediates, and strict avoidance of catabolism. Patients with UCD often require liver transplantation. However, preventing recurrence of hyperammonemia before liver transplantation can be difficult. Accordingly, there is a need in the art for improved compositions and methods for treating UCD.

Other metabolic disorders that affect the liver include the autosomal recessive disorder methylmalonic acidemia (MMA) (also called methylmalonic aciduria). MMA disrupts normal amino acid metabolism. Allotypes of methylmalonic acidemia cause defects in metabolic pathways that regulate the conversion of methylmalonyl-coenzyme A (CoA) to succinyl-CoA by the enzyme methylmalonyl-CoA mutase. This condition results in the inability to properly digest certain fats and proteins, thus accumulating toxic levels of methylmalonic acid in the blood. Methylmalonic acidemia alone is caused by alterations in one of the five genes: MMUT, MMAA, MMAB, MMADHC, or MCEE. Methylmalonic acidemia with homocystinuria is caused by mutations in the MMADHC, LMBRD1, and ABCD4 genes.

There is no specific treatment for methylmalonic acidemia. Treatment is currently limited to symptom management and includes aggressive treatment of decompensation events, special protein diets, vitamin B12 supplementation for vitamin B12-responsive subtypes, drug treatments such as carnitine, and decompensation events. Includes avoidance of stressors (such as fasting and illness). Liver or kidney transplantation (or both) has been shown to help some patients. These transplants provide the body with new cells that help break down methylmalonic acid normally.

Previous attempts to develop gene therapies for the treatment of inherited metabolic disorders, including those of the liver, have been plagued by the inability to achieve long-term expression of the delivered transgene in target tissues. . This problem is particularly pronounced in rapidly dividing tissues such as the liver of young adults. Existing gene therapy vectors, such as AAV vectors, suffer from the inability to integrate into the genome of the host, resulting in short-term expression of the introduced transgene. In response to this long-felt need in the art, the compositions and methods of the present disclosure provide transposon/transposase-based AAV vectors that provide long-term expression of delivered transgenes in target tissues. provide a possible solution.

SUMMARY The present disclosure provides an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in the 5' to 3' direction: a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3, b) SEQ ID NO: c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7; d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO:126; e) SEQ ID NO:22. f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97; g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8; h) the nucleic acid of SEQ ID NO:96 a second piggyBacITR sequence comprising sequences, i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:129, and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

The disclosure provides an AAVpiggyBac transposon polynucleotide comprising the nucleic acid sequence of SEQ ID NO:138.

The present disclosure provides an AAVpiggyBac transposon polynucleotide comprising, in a 5′ to 3′ direction, a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3, b) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:125. c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7; d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO:132; e) at least one comprising the nucleic acid sequence of SEQ ID NO:22. f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97; g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8; h) a second piggyBacITR comprising the nucleic acid sequence of SEQ ID NO:96. a sequence, i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:130, and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

The disclosure provides an AAVpiggyBac transposon polynucleotide comprising the nucleic acid sequence of SEQ ID NO:139.

The present disclosure provides an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in the 5' to 3' direction: a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3, b) SEQ ID NO:125. c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7; d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO:13; f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97; g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8; h) the nucleic acid sequence of SEQ ID NO:96. i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:131; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

The disclosure provides an AAVpiggyBac transposon polynucleotide comprising the nucleic acid sequence of SEQ ID NO:140.

The present disclosure provides an AAV transposase polynucleotide comprising, in a 5' to 3' direction, a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 127, b) a nucleic acid sequence of SEQ ID NO: 126. c) at least one transposase sequence comprising the nucleic acid sequence of SEQ ID NO:48; d) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:136; e) at least the nucleic acid sequence of SEQ ID NO:137. one DNA spacer sequence and f) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

The disclosure provides an AAV transposase polynucleotide comprising the nucleic acid sequence of SEQ ID NO:144.

The disclosure provides vectors comprising at least one of the AAVpiggyBac transposon polynucleotides of the disclosure. In some embodiments, the vector can be a viral vector. In some embodiments, the viral vector can be an AAV viral vector. In some aspects, the AAV viral vector can be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector. In some aspects, the AAV viral vector can be an AAV-KP-1 or AAV-NP59 viral vector. In some embodiments, the AAV viral vector can be an AAV-KP-1 viral vector.

The disclosure provides compositions comprising at least one vector of the disclosure.

The present disclosure provides a method of treating at least one metabolic liver disorder (MLD) in a subject in need thereof, comprising administering to the subject at least one of the disclosed polynucleotides, vectors, or compositions of the present disclosure. A method is provided comprising administering a therapeutically effective amount of

The present disclosure provides a method of treating at least one MLD in a subject in need thereof, comprising administering to the subject: a) an AAVpiggyBac transposon polynucleotide of the disclosure, or a vector comprising an AAVpiggyBac transposon polynucleotide of the disclosure, or Compositions, and b) methods comprising administering an AAV transposase polynucleotide of the disclosure, or a vector or composition comprising an AAV transposase polynucleotide of the disclosure.

The disclosure provides use of the polynucleotides, vectors, or compositions of the disclosure for the treatment of at least one MLD in a subject in need thereof, wherein the polynucleotide, vector, or composition is for administering at least one therapeutically effective amount to a subject.

The present disclosure provides a) AAVpiggyBac transposon polynucleotides of the disclosure, or a vector or composition comprising the AAVpiggyBac transposon polynucleotides of the disclosure, for use in the treatment of at least one MLD in a subject in need thereof; b) provide an AAV transposase polynucleotide of the disclosure, or in combination with a vector or composition comprising an AAV transposase polynucleotide of the disclosure.

In some embodiments, the at least one MLD is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate Synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperarginemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial hepatitis Intracholestasis type 1 (PFIC3), or any combination thereof. In some embodiments, the MLD is OTC deficiency.

Any of the above aspects can be combined with other aspects.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. By way of example, the terms "a," "an," and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. By way of example "element" means one or more elements. Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" may be used to refer to a stated element, integer or step or element. , integers, or groups of steps, but not to exclude any other elements, integers, or steps, or groups of elements, integers, or steps. About means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.5%, 1%, 0.5%, 0.1%, 0.5% of the stated value. 05%, or within 0.01%. All numerical values provided herein are modified by the term "about," unless otherwise clear from the context.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims that follow.

The above and further features will be more clearly understood from the following detailed description in conjunction with the accompanying drawings.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic representation of exemplary AAV transposase polynucleotides of the disclosure. FIG.

1 is a schematic representation of exemplary AAV transposase polynucleotides of the disclosure. FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

1 is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide of the present disclosure; FIG.

FIG. 10 is a graph showing BLI measured in mice treated with various viral vectors of the disclosure. FIG.

FIG. 10 is a graph showing BLI measured in mice treated with various concentrations of various viral vectors of the present disclosure. FIG.

FIG. 10 is a graph showing BLI measured in Otc ^spf-ash mice treated with various viral vectors of the disclosure. FIG.

2 is a series of graphs showing the amount of non-integrating vector copy number per diploid genome for various viral vectors of the present disclosure administered to Otc ^spf-ash mice.

2 is a series of graphs showing the amount of non-integrated vector copy number per diploid genome and integrated vector copy number per diploid genome for various viral vectors of the present disclosure administered to Otc ^spf-ash mice. .

2 is a series of graphs showing amounts of human OTC mRNA and SPB mRNA relative to levels of mouse OTC mRNA in Otc ^spf-ash mice treated with viral vectors of the present disclosure.

FIG. 10 is a graph showing the correlation of human OTC mRNA or SPB mRNA to total vector copy number per diploid genome in Otc ^spf-ash mice treated with viral vectors of the present disclosure.

FIG. 10 is a graph showing survival probabilities in an induced hyperammonemia mouse model treated with a viral vector of the present disclosure. FIG.

FIG. 10 is a graph showing ammonia concentration in plasma obtained from an induced hyperammonemia mouse model treated with a viral vector of the present disclosure. FIG.

FIG. 10 is a graph showing liver BLI measured in mice treated with various viral vectors of the disclosure. FIG.

FIG. 10 is a graph showing the amount of human OTC mRNA versus the level of mouse OTC mRNA in mice treated with viral vectors of the present disclosure. FIG.

FIG. 10 is a graph showing the amount of SBP mRNA versus mouse OTC mRNA levels in mice treated with viral vectors of the present disclosure. FIG.

FIG. 10 is a graph showing the amount of human OTC protein versus mouse OTC protein levels in mice treated with a viral vector of the present disclosure. FIG.

FIG. 10 is a graph showing BLI measured in mice treated with various viral vectors of the disclosure. FIG.

FIG. 10 is a graph showing the amount of human OTC mRNA versus the level of mouse OTC mRNA in mice treated with viral vectors of the present disclosure. FIG.

FIG. 10 is a graph showing the amount of SBP mRNA versus mouse OTC mRNA levels in mice treated with viral vectors of the present disclosure. FIG.

FIG. 10 is a graph showing the amount of human OTC protein versus mouse OTC protein levels in mice treated with a viral vector of the present disclosure. FIG.

FIG. 4 shows immunohistochemical analysis of hepatocytes isolated from mice treated with viral vectors of the present disclosure.

DETAILED DESCRIPTION The present disclosure provides compositions and methods for the treatment of metabolic liver disorders, including but not limited to urea cycle disorders (UCD). This composition and method are described in further detail herein.

Disclosed composition

Adeno-associated virus (AAV) piggyBac transposon polynucleotides

The present disclosure provides compositions comprising adeno-associated virus (AAV) piggyBac transposon polynucleotides.

In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one AAV inverted terminal repeat (ITR) sequence. In some aspects, an AAV piggyBac transposon polynucleotide can comprise at least one piggyBacITR sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one insulator sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can include at least one promoter sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one transgene sequence. In some aspects, the AAV piggyBac transposon polynucleotide can comprise at least one poly A sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one self-cleaving peptide sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can include at least one DNA spacer sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one Int6F sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one Int6P1 sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one Int6R sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one JctR sequence. In some aspects, the AAVpiggyBac transposon polynucleotide can comprise at least one MCS sequence.

The AAVpiggyBac transposon polynucleotide can comprise a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by a second insulator sequence followed by a second piggyBacITR sequence, followed by a second AAVITR sequence.

The AAVpiggyBac transposon polynucleotide can comprise a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence; Here, between the first insulator sequence and the second insulator sequence are at least one promoter sequence, at least one transgene sequence, at least one self-cleaving peptide sequence, and at least one poly A sequence. Any combination exists.

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequences wherein, between the first insulator sequence and the second insulator sequence, at least one promoter sequence, at least one transgene sequence, at least one self-cleaving peptide sequence, and at least one poly A sequence. .

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by a second insulator sequence followed by a second piggyBacITR followed by a second AAVITR sequence, wherein between the first insulator sequence and the second insulator sequence are at least one promoter sequence, at least one transgene sequence, Any combination of at least one self-cleaving peptide sequence and at least one poly A sequence is present.

The AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a poly A sequence, a second insulator sequence, a first 2 piggyBacITR sequences, and a second AAVITR sequence.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene A poly A sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by at least one promoter sequence followed by at least one transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, followed by a second AAVITR sequence.

In the non-limiting examples of AAVpiggyBac transposon polynucleotides described above, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and at least one transgene sequence encodes a methylmalonyl-CoA mutase (MUT1) polypeptide. Encoding nucleic acid sequences can be included. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

The AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a poly A sequence, a second insulator sequence, a first 2 piggyBacITR sequences, at least one DNA spacer sequence, and a second AAVITR sequence.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene A poly A sequence, a second insulator sequence, a second piggyBacITR sequence, at least one DNA spacer sequence, and a second AAVITR sequence.

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by at least one promoter sequence followed by at least one transgene sequence, followed by a poly A sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, followed by at least one DNA spacer sequence, and followed by a second AAVITR sequence.

In the non-limiting examples of AAV piggyBac transposon polynucleotides described above, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and at least one transgene sequence encodes an ornithine transcarbamylase (OTC) polypeptide. can include a nucleic acid sequence that This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG. 3A.

In some embodiments, the AAVpiggyBac transposon polynucleotide can include at least one DNA spacer sequence between the second piggyBacITR sequence and the second AAVITR sequence.

The AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a poly A sequence, a second insulator sequence, a first 2 piggyBacITR sequences, a second AAVITR sequence, and at least one DNA spacer sequence.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene A poly A sequence, a second insulator sequence, a second piggyBacITR sequence, a second AAVITR sequence, and at least one DNA spacer sequence.

In some embodiments, the AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by at least one promoter sequence followed by at least one transgene sequence, followed by a poly A sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, followed by a second AAVITR sequence, followed by at least one DNA spacer sequence.

In the non-limiting examples of AAV piggyBac transposon polynucleotides described above, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and at least one transgene sequence encodes an ornithine transcarbamylase (OTC) polypeptide. can include a nucleic acid sequence that This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in Figure 3B.

In some embodiments, the AAVpiggyBac transposon polynucleotide can include at least one DNA spacer after the second AAVITR sequence. A non-limiting example of an AAVpiggyBac transposon polynucleotide having at least one DNA spacer after the second AAVITR sequence is shown in FIG. 3B.

The AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, at least one self-cleaving peptide sequence, at least a second A transgene sequence, a poly A sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence can be included.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, at least one self-cleaving peptide sequence, at least a second transgene sequence, a poly A sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by at least one promoter sequence followed by a first transgene sequence, followed by at least one self-cleaving peptide sequence, followed by at least a second transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, followed by a second AAVITR sequences of

In the non-limiting examples of AAVpiggyBac transposon polynucleotides described above, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and the first transgene sequence encodes an ornithine transcarbamylase (OTC) polypeptide. at least one self-cleaving peptide sequence can comprise one encoding a T2A peptide and at least a second transgene sequence encoding an ornithine transcarbamylase (OTC) polypeptide can include a nucleic acid sequence that This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

In another non-limiting example of the aforementioned AAVpiggyBac transposon polynucleotide, at least one promoter sequence can comprise a thyroxine binding globulin (TBG) promoter and the first transgene sequence comprises an ornithine transcarbamylase (OTC) poly nucleic acid sequences encoding peptides, at least one self-cleaving peptide sequence encoding a T2A peptide, and at least a second transgene sequence comprising a luciferase sequence (e.g., NanoLuc) can be done. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

In the non-limiting examples of AAVpiggyBac transposon polynucleotides described above, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and the first transgene sequence encodes an ornithine transcarbamylase (OTC) polypeptide. at least one self-cleaving peptide sequence encoding a T2A peptide; and at least a second transgene sequence encoding an inducible caspase-9 (iCAS9) polypeptide. Encoding nucleic acid sequences can be included. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

The AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a first promoter sequence, a first transgene sequence, at least a second promoter sequence, at least a second transgene sequence. A gene sequence, a poly A sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence can be included.

The AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a first promoter sequence, a first transgene sequence, at least a second A promoter sequence, at least a second transgene sequence, a poly A sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence can be included.

The AAVpiggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by a first promoter sequence followed by a first transgene sequence followed by at least a first 2 promoter sequences, followed by at least a second transgene sequence, followed by a poly A sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, followed by a second AAVITR sequence. can.

In the non-limiting examples of AAVpiggyBac transposon polynucleotides described above, the first promoter sequence can comprise a hybrid liver promoter (HLP) and the first transgene sequence encodes an ornithine transcarbamylase (OTC) polypeptide. at least a second promoter sequence comprising an HLP, and at least a second transgene sequence comprising a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide can be done. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

In some aspects, an AAVpiggyBac transposon polynucleotide can comprise more than one transgene sequence. In some embodiments where the AAVpiggyBac transposon polynucleotide comprises more than one transgene sequence, individual transgene sequences can be separated by self-cleaving peptide sequences. In some embodiments in which the AAVpiggyBac transposon polynucleotide comprises more than one self-cleaving peptide sequence, the self-cleaving peptide sequences can be the same or different.

In some aspects, an AAVpiggyBac transposon polynucleotide can comprise more than one transgene sequence. In some embodiments where the AAVpiggyBac transposon polynucleotide contains more than one transgene sequence, the AAVpiggyBac transposon can contain multiple copies of a nucleic acid sequence encoding the same polypeptide. In a non-limiting example, the AAVpiggyBac transposon polynucleotide can include a first transgene sequence and a second transgene sequence, wherein the first transgene sequence and the second transgene sequence are ornithine transgene sequences. A nucleic acid encoding a carbamylase (OTC) polypeptide is included.

In some aspects, an AAVpiggyBac transposon polynucleotide can comprise more than one promoter sequence. In some embodiments in which the AAVpiggyBac transposon polynucleotide comprises more than one promoter sequence, the promoter sequences can be the same or different.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises: a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, a first self-cleaving peptide a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a poly A sequence, a second insulator sequence, and a second AAVITR sequence.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene a sequence, a first self-cleaving peptide sequence, a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a poly A sequence, a second insulator sequence, and a second AAVITR Can contain arrays.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by at least one promoter sequence followed by a first transgene sequence, followed by a first self-cleavage peptide sequence, followed by a second transgene sequence, followed by at least a second self-cleavage peptide sequence, followed by at least a third transgene sequence, followed by a poly A sequence, followed by A second insulator sequence can be included followed by a second AAVITR sequence.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises: a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, a first self-cleaving peptide a sequence, a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a poly A sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence. can contain.

In some embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence first self-cleaving peptide sequence second transgene sequence at least second self-cleaving peptide sequence at least third transgene sequence poly A sequence second insulator sequence second piggyBacITR sequence , and a second AAVITR sequence

In some embodiments, the AAV piggyBac transposon polynucleotide comprises a first AAVITR sequence followed by a first piggyBacITR sequence followed by a first insulator sequence followed by at least one promoter sequence followed by a first transgene sequence, followed by a first self-cleavage peptide sequence, followed by a second transgene sequence, followed by at least a second self-cleavage peptide sequence, followed by at least a third transgene sequence, followed by a poly A sequence, followed by A second insulator sequence can be included, followed by a second piggyBacITR sequence, followed by a second AAVITR sequence.

In the non-limiting examples of AAVpiggyBac transposon polynucleotides described above, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and the first transgene sequence encodes an ornithine transcarbamylase (OTC) polypeptide. The second transgene sequence can comprise a fluorescent protein sequence (eg GFP or eGFP) and at least the third transgene sequence can comprise a luciferase sequence (eg NanoLuc). In this non-limiting example, both the first self-cleavage peptide sequence and the at least second self-cleavage peptide sequence can comprise a nucleic acid sequence encoding a T2A peptide or a GSG-T2A peptide. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

In another non-limiting example of the AAVpiggyBac transposon polynucleotides described above, at least one promoter sequence can comprise an LP1 promoter, and the first transgene sequence is a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. wherein the second transgene sequence can comprise a fluorescent protein sequence (eg GFP or eGFP) and at least the third transgene sequence can comprise a luciferase sequence (eg NanoLuc). In this non-limiting example, both the first self-cleaving peptide sequence and the at least second self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a T2A peptide or a GSG-T2A peptide. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG.

In some embodiments, the AAVpiggyBac transposon polynucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% %, 99%, or 100% (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are identical.

AAVITR sequence

In some aspects, the AAVITR sequence can include any AAVITR sequence known in the art. In some embodiments, the AAVITR sequence is at least 65%, 70%, 75%, 80%, 85%, 90% with any of the sequences set forth in SEQ ID NOs: 1-4, 93-94, 105-106, and 127. %, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprise, consist essentially of, or consist of nucleic acid sequences that are .

In some embodiments, the first AAVITR sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second AAVITR sequence is at least 65% identical to SEQ ID NO:2 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first AAVITR sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% SEQ ID NO:3 or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second AAVITR sequence is at least 65% identical to SEQ ID NO:4 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first AAVITR sequence is SEQ ID NO: 93 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second AAVITR sequence is at least 65% identical to SEQ ID NO:94 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first AAVITR sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO: 105 or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second AAVITR sequence is at least 65% identical to SEQ ID NO: 106 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first AAVITR sequence is SEQ ID NO: 127 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second AAVITR sequence is at least 65% identical to SEQ ID NO:4 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

piggyBac ITR sequence

In some embodiments, the piggyBacITR sequence can comprise any piggyBacITR sequence known in the art. 96, and 125 with at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% ( or any percentage therebetween) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some embodiments, the first piggyBacITR sequence is SEQ ID NO: 5 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second piggyBacITR sequence is at least 65% identical to SEQ ID NO:6 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first piggyBacITR sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second piggyBacITR sequence is at least 65% identical to SEQ ID NO:5 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first piggyBacITR sequence is SEQ ID NO: 95 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second piggyBacITR sequence is at least 65% identical to SEQ ID NO:96 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some embodiments, the first piggyBacITR sequence is SEQ ID NO: 125 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or consists essentially of or consists of a nucleic acid sequence that is 100% (or any percentage therebetween) identical, and the second piggyBacITR sequence is at least 65% identical to SEQ ID NO:96 , 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) may consist essentially of or consist of

In some aspects of the methods of the present disclosure, the piggyBacITR sequences within the AAV piggyBac transposon, such as the first piggyBacITR sequence and/or the second piggyBacITR sequence, are the Sleeping Beauty transposon ITR, Helraiser. It may comprise, consist essentially of, or consist of a transposon ITR, a Tol2 transposon ITR, a TcBuster transposon ITR, or any combination thereof.

In some aspects, the piggyBacITR sequences of the present disclosure can be flanked on either or both ends by at least one of the following sequences: 5'-CTAA-3', 5'-TTAG-3 ', 5'-ATAA-3', 5'-TCAA-3', 5'-AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5′-TTTA-3′, 5′-TTAC-3′, 5′-ACTA-3′, 5′-AGGG-3′, 5′-CTAG-3′, 5′-TGAA-3′, 5′ -AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'-TGAA-3', 5'-AAAT -3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3 ', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5′-GTCC-3′, 5′-TAAG-3′, 5′-TCTA-3′, 5′-TGAG-3′, 5′-TGTT-3′, 5′-TTCA-3′, 5′ -TTCT-3', and 5'-TTTT-3'. In some embodiments, the piggyBacITR sequence can be flanked by 5'-TTAA-3'. Accordingly, any AAV transposase polynucleotide, AAV piggyBac transposon polynucleotide, and/or any liver nanoplasmid of the present disclosure can further comprise any one of the following: 5' flanking the piggyBacITR sequence; -CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'-AGTT-3', 5'-ATTA-3', 5'-GTTA -3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3 ', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5′-TGAA-3′, 5′-AAAT-3′, 5′-AATC-3′, 5′-ACAA-3′, 5′-ACAT-3′, 5′-ACTC-3′, 5′ -AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA -3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3 ', 5'-TTCA-3', 5'-TTCT-3', 5'-TTTT-3'.

Insulator array

In some embodiments, the insulator sequence can comprise any insulator sequence known in the art. In some embodiments, the insulator sequence is at least 65%, 70%, 75%, 80%, 85%, 90% with any of the sequences set forth in SEQ ID NOS: 7-8, 77-80, and 91-92. %, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprise, consist essentially of, or consist of nucleic acid sequences that are .

In some embodiments, the first insulator sequence is SEQ ID NO: 7 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% %, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of a nucleic acid sequence that is identical to, and the second insulator sequence is at least A nucleic acid sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to may comprise, consist essentially of, or consist of;

In some embodiments, the first insulator sequence is SEQ ID NO: 77 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% %, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of a nucleic acid sequence that is identical to, and the second insulator sequence is at least A nucleic acid sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to may comprise, consist essentially of, or consist of;

In some embodiments, the first insulator sequence is SEQ ID NO: 79 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% %, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of a nucleic acid sequence that is identical to, and the second insulator sequence is at least A nucleic acid sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to may comprise, consist essentially of, or consist of;

In some embodiments, the first insulator sequence is SEQ ID NO: 91 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% %, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of a nucleic acid sequence that is identical, and the second insulator sequence is at least A nucleic acid sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to may comprise, consist essentially of, or consist of;

promoter sequence

In some embodiments, the promoter sequence can comprise any promoter sequence known in the art. In some embodiments, the promoter sequence can include any liver-specific promoter sequence known in the art.

In some embodiments, the promoter sequence is any of the sequences set forth in SEQ ID NOs: 9-16, 69, 107, 126, 132, 145, and 146 and at least 65%, 70%, 75%, 80%, 85% %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of nucleic acid sequences that are be able to.

In some aspects, the promoter sequence can comprise a hybrid liver promoter (HLP). HLP is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% with SEQ ID NO: 9, 107, or 126 It can comprise, consist essentially of, or consist of (or any percentage in between) identical nucleic acid sequences.

In some aspects, the promoter sequence can comprise the LP1 promoter. LP1 promoter is SEQ ID NO: 10 or 132 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%%, or 100% ( or any percentage therebetween) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some embodiments, the promoter sequence can include leukocyte-specific expression of the pp52 (LSP1) long promoter. The LSP1 long promoter comprises SEQ ID NO: 11 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage of) comprises, consists essentially of, or consists of identical nucleic acid sequences.

In some aspects, the promoter sequence can include a thyroxine binding globulin (TBG) promoter. The TBG promoter comprises SEQ ID NO: 12 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some aspects, the promoter sequence can comprise the wTBG promoter. The wTBG promoter comprises SEQ ID NO: 13 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some aspects, the promoter sequence can include the liver combinatorial bundle (HCB) promoter. The HCB promoter comprises SEQ ID NO: 14 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some embodiments, the promoter sequence can include the 2xApoE-hAAT promoter. 2xApoE-hAAT promoter is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some embodiments, the promoter sequence can comprise leukocyte-specific expression of the pp52(LSP1)+chimeric intron promoter. The LSP1+ chimeric intron promoter is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some embodiments, the promoter sequence can include the cytomegalovirus (CMV) promoter. The CMV promoter comprises SEQ ID NO: 69 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some aspects, the promoter sequence can include the TTR promoter. The TTR promoter comprises SEQ ID NO: 145 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

In some aspects, the promoter sequence can comprise the TTRm promoter. The TTRm promoter comprises SEQ ID NO: 146 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or in between) any percentage) comprising, consisting essentially of, or consisting of nucleic acid sequences that are identical.

transgene sequence

In some embodiments, the transgene sequence can comprise a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide. In some embodiments, the transgene sequence can comprise a nucleic acid sequence encoding a MUT1 polypeptide, wherein the MUT1 polypeptide is at least 65%, 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences, or essentially from consist of or consist of In some embodiments, the acid sequence encoding the MUT1 polypeptide is at least 65%, 70%, 75%, 80%, 85%, comprising, consisting essentially of, or consisting of nucleic acid sequences that are 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical can.

In some aspects, the transgene sequence can comprise a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. In some embodiments, the transgene sequence can comprise a nucleic acid sequence encoding an OTC polypeptide, wherein the OTC polypeptide is SEQ ID NO: 21, 81, 123, or 124 and at least 65%, 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences, or essentially from consist of or consist of In some embodiments, the acid sequence encoding the OTC polypeptide is at least 65%, 70%, 75%, 80%, 85%, comprising, consisting essentially of, or consisting of nucleic acid sequences that are 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical can.

In some embodiments, a transgene sequence can comprise a nucleic acid sequence encoding an iCAS9 polypeptide. In some embodiments, the transgene sequence can comprise a nucleic acid sequence encoding an iCAS9 polypeptide, wherein the iCAS9 polypeptide is SEQ ID NO: 24 or 84 and at least 65%, 70%, 75%, 80% comprises or consists essentially of nucleic acid sequences that are %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical; or consists of In some embodiments, the acid sequence encoding the iCAS9 polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% any of the sequences set forth in SEQ ID NO: 25 or 85. , 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences.

In some embodiments, transgene sequences can be codon optimized according to methods known in the art.

In some embodiments, a nucleic acid sequence encoding a polypeptide (eg, OTC, MUT1, etc.) can be a codon-optimized nucleic acid sequence encoding the polypeptide. A codon-optimized nucleic acid sequence encoding a polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% different from a wild-type human nucleic acid sequence encoding a polypeptide. %, 98%, 99% (or any percentage therebetween) or less that comprise, consist essentially of, or consist of nucleic acid sequences that are identical.

SEQ ID NOs: 19, 20, 22, 23, 82, and 83 are unique codon-optimized nucleic acid sequences that can be included in the polynucleotides, vectors and compositions of the present disclosure.

In some embodiments, codon-optimized nucleic acid sequences encoding polypeptides, such as those set forth in SEQ ID NOS: 19, 20, 22, 23, 82, and 83, may be free of donor splice junctions. In some embodiments, the codon-optimized nucleic acid sequence encoding the polypeptide is about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or It can contain about 9, or about 10 or less donor splice sites. In some embodiments, the codon-optimized nucleic acid sequence encoding the polypeptide is at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 fewer donor splice sites. Without wishing to be bound by theory, removal of donor splice sites in codon-optimized nucleic acid sequences unexpectedly and unexpectedly increases expression of the polypeptide in vivo because cryptic splicing is prevented. Sometimes. Furthermore, cryptic splicing can vary between different subjects, which suggests that expression levels of polypeptides containing donor splice sites can vary unpredictably between different subjects. means.

In some embodiments, the codon-optimized nucleic acid sequences encoding the polypeptides, such as set forth in SEQ ID NOS: 19, 20, 22, 23, 82, and 83, are sequences of wild-type human nucleic acid sequences encoding the polypeptides. may have a GC content different from the GC content of In some embodiments, the GC content of a codon-optimized nucleic acid sequence encoding a polypeptide is more evenly distributed throughout the nucleic acid sequence as compared to a wild-type human nucleic acid sequence encoding a polypeptide. While not wishing to be bound by theory, by more evenly distributing GC content throughout the nucleic acid sequence, codon-optimized nucleic acid sequences exhibit a more uniform melting temperature ("Tm ”). Uniformity of melting temperatures unexpectedly increases expression of codon-optimized nucleic acids in human subjects because transcription and/or translation of nucleic acid sequences occurs with less stalling of the polymerase and/or ribosomes.

In some embodiments, the codon-optimized nucleic acid sequences encoding the polypeptides, such as those set forth in SEQ ID NOs: 19, 20, 22, 23, 82, and 83, are wild-type or codon-optimized polypeptide-encoding sequences. at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300% in human subjects compared to the untreated nucleic acid sequence , at least 500%, or at least 1000% increased expression.

In some embodiments, at least one transgene sequence can be operably linked to at least one promoter sequence present on the same polynucleotide.

Poly A sequence

In some embodiments, the poly A sequence can comprise any poly A sequence known in the art. Non-limiting examples of poly A sequences include, but are not limited to, SV40 poly A sequences. In some embodiments, the poly A sequence is at least 65%, 70%, 75%, 80%, 85%, 90% with any of the sequences set forth in SEQ ID NOS: 26-27, 97, 108, 128, and 136. %, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprise, consist essentially of, or consist of nucleic acid sequences that are .

self-cleaving peptide sequence

In some embodiments, the self-cleaving peptide sequence can comprise any self-cleaving peptide sequence known in the art. In some embodiments, the self-cleavage peptide sequence can comprise a 2A self-cleavage peptide sequence known in the art. Non-limiting examples of self-cleaving peptides include T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide.

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a T2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding a T2A peptide, wherein the T2A peptide is at least 65%, 70%, 75%, 80%, 85%, 90% of SEQ ID NO:28. , 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprise, consist essentially of, or consist of nucleic acid sequences that are 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-T2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding a GSG-T2A peptide, wherein the GSG-T2A peptide is at least 65%, 70%, 75%, 80%, 85% of SEQ ID NO:29. %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of nucleic acid sequences that are . In some embodiments, the nucleic acid sequences encoding the GSG-T2A peptides are SEQ ID NOs: 30-32 and 135 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of nucleic acid sequences that are 97%, 98%, 99%, or 100% identical.

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding an E2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding an E2A peptide, wherein the E2A peptide is at least 65%, 70%, 75%, 80%, 85%, 90% SEQ ID NO:33 , 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprise, consist essentially of, or consist of nucleic acid sequences that are 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-E2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding a GSG-E2A peptide, wherein the GSG-E2A peptide is at least 65%, 70%, 75%, 80%, 85% of SEQ ID NO:34. %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of nucleic acid sequences that are .

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding an F2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding an F2A peptide, wherein the F2A peptide is at least 65%, 70%, 75%, 80%, 85%, 90% of SEQ ID NO:35 , 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprise, consist essentially of, or consist of nucleic acid sequences that are 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-F2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding a GSG-F2A peptide, wherein the GSG-F2A peptide is at least 65%, 70%, 75%, 80%, 85% of SEQ ID NO:36. %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of nucleic acid sequences that are .

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a P2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding a P2A peptide, wherein the P2A peptide is at least 65%, 70%, 75%, 80%, 85%, 90% of SEQ ID NO:37 , 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprise, consist essentially of, or consist of nucleic acid sequences that are 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, a self-cleaving peptide sequence can comprise a nucleic acid sequence encoding a GSG-P2A peptide. In some embodiments, the self-cleaving peptide sequence comprises a nucleic acid sequence encoding a GSG-P2A peptide, wherein the GSG-P2A peptide is at least 65%, 70%, 75%, 80%, 85% of SEQ ID NO:38. %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprises, consists essentially of, or consists of nucleic acid sequences that are .

DNA spacer sequence

In some embodiments, the DNA spacer sequence is at least 65%, 70%, 75%, 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences.

The DNA spacer sequence can be located anywhere within the AAVpiggyBac transposon polynucleotide or AAVpiggyBac transposase polynucleotide.

Int6F sequence

In some embodiments, the Int6F sequence is SEQ ID NO: 98 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are identical. In some embodiments, the Int6F sequence can be located between the polyA sequence and the second insulator sequence.

Int6P1 sequence

In some embodiments, the Int6P1 sequence is SEQ ID NO:99 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are identical. In some embodiments, the IntP1 sequence can be located between the polyA sequence and the second insulator sequence.

Int6R sequence

In some embodiments, the Int6R sequence is SEQ ID NO: 100 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are identical. In some embodiments, the Int6R sequence can be located between the poly A sequence and the second insulator sequence.

JctR sequence

In some embodiments, the JctR sequence is SEQ ID NO: 101 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are identical. In some embodiments, the JctR sequence can be located between the second piggyBacITR sequence and the second AAVITR sequence.

In some embodiments, the AAVpiggyBac transposon polynucleotide is SEQ ID NO: 138 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or may comprise, consist essentially of, or consist of nucleic acid sequences that are 100% (or any percentage therebetween) identical.

In some embodiments, the AAVpiggyBac transposon polynucleotide is SEQ ID NO: 139 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or may comprise, consist essentially of, or consist of nucleic acid sequences that are 100% (or any percentage therebetween) identical.

In some embodiments, the AAVpiggyBac transposon polynucleotide is SEQ ID NO: 140 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or may comprise, consist essentially of, or consist of nucleic acid sequences that are 100% (or any percentage therebetween) identical.

In some embodiments, the AAVpiggyBac transposon polynucleotide is SEQ ID NO: 141 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or may comprise, consist essentially of, or consist of nucleic acid sequences that are 100% (or any percentage therebetween) identical.

In some embodiments, the AAVpiggyBac transposon polynucleotide is SEQ ID NO: 142 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or may comprise, consist essentially of, or consist of nucleic acid sequences that are 100% (or any percentage therebetween) identical.

In some embodiments, the AAVpiggyBac transposon polynucleotide is SEQ ID NO: 143 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or may comprise, consist essentially of, or consist of nucleic acid sequences that are 100% (or any percentage therebetween) identical.

MCS array

In some embodiments, the MCS sequence is SEQ ID NO: 102 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are identical. In some embodiments, the MCS sequence can be located between the second piggyBacITR sequence and the second AAVITR sequence.

AAV transposase polynucleotide

The present disclosure provides compositions comprising AAV transposase polynucleotides.

In some aspects, the AAV transposase polynucleotide can comprise at least one AAV inverted terminal repeat (ITR) sequence. In some aspects, an AAV transposase polynucleotide can comprise at least one promoter sequence. In some aspects, an AAV transposase polynucleotide can comprise at least one transposase sequence. In some aspects, an AAV transposon polynucleotide can comprise at least one poly A sequence. In some embodiments, AAV transposon polynucleotides can include at least one DNA spacer sequence.

In some aspects, an AAV transposase polynucleotide can comprise a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, and a second AAVITR sequence.

In some aspects, the AAV transposase polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, and a second AAVITR sequences can be included.

In some embodiments, the AAV transposase polynucleotide comprises a first AAVITR sequence, followed by at least one promoter sequence, followed by at least one transposase sequence, followed by a poly A sequence, followed by a second AAVITR sequences can be included.

In some embodiments, the AAV transposase polynucleotide comprises a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, at least one DNA spacer sequence, and a second AAVITR Can contain arrays.

In some aspects, the AAV transposase polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, at least one DNA A spacer sequence and a second AAVITR sequence can be included.

In some embodiments, the AAV transposase polynucleotide comprises a first AAVITR sequence followed by at least one promoter sequence followed by at least one transposase sequence followed by a poly A sequence followed by at least one DNA A spacer sequence followed by a second AAVITR sequence can be included.

In the foregoing non-limiting examples of AAV transposase polynucleotides, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and at least one transposase sequence can comprise a SuperpiggyBac™ (SPB) transposase sequence. A nucleic acid sequence encoding a sase polypeptide can be included. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in FIG. 4A.

In some embodiments, the AAV transposase polynucleotide can include at least one DNA spacer sequence between the poly A sequence and the second AAVITR sequence, as shown in the non-limiting example of Figure 4A. can.

In some embodiments, the AAV transposase polynucleotide comprises a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, a second AAVITR sequence, and at least one DNA spacer Can contain arrays.

In some aspects, the AAV transposase polynucleotide comprises, from 5′ to 3′, a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, a second AAVITR sequence, and at least one DNA spacer sequence.

In some embodiments, the AAV transposase polynucleotide comprises a first AAVITR sequence followed by at least one promoter sequence followed by at least one transposase sequence followed by a poly A sequence followed by a second AAVITR sequence, followed by at least one DNA spacer sequence.

In the foregoing non-limiting examples of AAV transposase polynucleotides, at least one promoter sequence can comprise a hybrid liver promoter (HLP) and at least one transposase sequence can comprise SuperpiggyBac™ (SPB) A nucleic acid sequence encoding a transposase polypeptide can be included. This non-limiting example of an AAVpiggyBac transposon polynucleotide is shown in Figure 4B.

In some embodiments, the AAV transposase polynucleotide can include at least one DNA spacer sequence after the second AAVITR sequence, as shown in the non-limiting example of Figure 4B.

In some embodiments, the AAV transposase polynucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99%, or 100% (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are 100% identical.

In some embodiments, the AAV transposase polynucleotide has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99%, or 100% (or any percentage therebetween) can comprise, consist essentially of, or consist of nucleic acid sequences that are 100% identical.

transposase sequence

In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding any transposase polypeptide known in the art. In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a piggyBac™ (PB) transposase polypeptide. In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a piggyBac-like (PBL) transposase polypeptide. In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a SuperpiggyBac™ (SPB) transposase polypeptide.

Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are US Pat. No. 6,218,182, US Pat. No. 6,962,810. It is described in detail in US Pat. No. 8,399,643 and PCT Publication No. WO2010/099296.

PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of transposons and allocate their contents to the sequence 5′-TTAA- in a chromosomal site (TTAA target sequence). Inserted between 3′ ITRs. The target sequences of PB or PBL transposons are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'-AGTT-3', 5 '-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'- AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA- 3′, 5′-TCTC-3′, 5′-TGAA-3′, 5′-AAAT-3′, 5′-AATC-3′, 5′-ACAA-3′, 5′-ACAT-3′ , 5′-ACTC-3′, 5′-AGTG-3′, 5′-ATAG-3′, 5′-CAAA-3′, 5′-CACA-3′, 5′-CATA-3′, 5 '-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'- TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system has no payload limitations for genes of interest that can be included between ITRs.

Exemplary amino acid sequences of one or more of PB, PBL, and SPB transposases are found in US Pat. No. 6,218,185, US Pat. No. 6,962,810, and US Pat. disclosed in No. In preferred embodiments, the PB transposase is SEQ ID NO: 39 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage in between) identical amino acid sequences.

The PB or PBL transposase comprises an amino acid sequence having amino acid substitutions at two or more, three or more, or each of positions 30, 165, 282, and/or 538 of the sequence of SEQ ID NO:39, or can consist of The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of the sequence of SEQ ID NO: 39, wherein the amino acid substitution of the sequence at position 30 is isoleucine (I) for valine (V) , the amino acid substitution at position 165 may be a substitution of glycine (G) for serine (S), the amino acid substitution at position 282 may be a substitution of methionine (M) for valine (V), and the amino acid substitution at position 538 may be Substitutions may be replacement of asparagine (N) by lysine (K). In preferred embodiments, the SPB transposase is SEQ ID NO: 40 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % (or any percentage in between) identical amino acid sequences.

In certain embodiments, the transposase comprises the above mutations at positions 30, 165, 282, and/or 538, the PB, PBL, and SPB transposases are 3, 46 , 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340 , 421, 436, 456, 470, 486, 503, 552, 570, and 591, which are disclosed in PCT Publication Nos. WO2019/173636 and PCT/US2019/ 049816 in detail.

In preferred embodiments, the PB transposase is SEQ ID NO: 41 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage in between) identical amino acid sequences.

The PB or PBL transposase comprises or consists of an amino acid sequence having amino acid substitutions at two or more, three or more, or each of positions 29, 164, 281, and/or 537 of the sequence of sequence 41. obtain. The transposase may be a SPB transposase comprising or consisting of the amino acid sequence of the sequence of SEQ ID NO: 41, wherein the amino acid substitution at position 29 may be replacement of isoleucine (I) with valine (V), 164 The amino acid substitution at position 281 may be a substitution of glycine (G) for serine (S), the amino acid substitution at position 281 may be a substitution of methionine (M) for valine (V), and the amino acid substitution at position 537 may be a substitution of lysine (K ) may be substituted for asparagine (N). In preferred embodiments, the SPB transposase is SEQ ID NO: 42 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % (or any percentage in between) identical amino acid sequences.

In certain embodiments, the transposase comprises the above mutations at positions 29, 164, 281, and/or 537, the PB, PBL, and SPB transposases are 2, 45 , 81, 102, 118, 124, 176, 179, 184, 186, 199, 206, 208, 225, 234, 239, 240, 242, 257, 295, 297, 310, 314, 318, 326, 327, 339 , 420, 435, 455, 469, 485, 502, 551, 569, and 590, which are disclosed in PCT Publication Nos. WO2019/173636 and PCT/US2019/ 049816 for further details.

PB, PBL, or SPB transposases are isolated or derived from insects, vertebrates, crustaceans, or urochordates, as described in more detail in PCT Publication Nos. WO2019/173636 and PCT/US2019/049816. can do. In preferred embodiments, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or the silkworm (Bombyx mori) (GenBank Accession No. BAD11135).

A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In preferred embodiments, the overactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of overactive PB or PBL transposases are disclosed in US Pat. No. 6,218,185, US Pat. No. 6,962,810, US Pat. No. 8,399,643, and WO2019/173636. there is A list of hyperactive amino acid substitutions is disclosed in US Pat. No. 10,041,077.

In some embodiments, the PB, PBL, or SPB transposase is integration defective. An integration-deficient PB, PBL, or SPB transposase is a transposase that can excise the corresponding transposon, but integrates the excised transposon less frequently than the corresponding wild-type transposase. Examples of integration deficient PB, PBL, or SPB transposases are found in US Pat. No. 6,218,185, US Pat. No. 6,962,810, US Pat. disclosed. A list of incorporated defective amino acid substitutions is disclosed in US Pat. No. 10,041,077.

In some aspects, the PB, PBL, or SPB transposase can be fused to a nuclear localization signal. Examples of PB, PBL, or SPB transposases fused to nuclear localization signals are described in US Pat. No. 6,218,185, US Pat. No. 6,962,810, US Pat. No. 8,399,643 and WO2019/173636. The nuclear localization signal is SEQ ID NO: 43 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) comprising, consisting essentially of, or consisting of identical amino acid sequences. The nuclear localization signal is SEQ ID NO: 44 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or Any percentage therebetween can be encoded by a nucleic acid sequence comprising, consisting essentially of, or consisting of a nucleic acid sequence that is identical.

In some embodiments, the nuclear localization signal can be fused to the PB, PBL, or SPB transposase using a G4S linker located between the NLS and the PB, PBL, or SPB. . The G4S linker is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any in between) of SEQ ID NO:45 (percentage of) identical amino acid sequences. The G4S linker is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any in between) of SEQ ID NO:46 percentage of the identity) can be encoded by a nucleic acid sequence comprising, consisting essentially of, or consisting of an identical nucleic acid sequence.

In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding an SBP transposase polypeptide fused to an NLS, wherein the SBP transposase polypeptide fused to the NLS is SEQ ID NO: is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to 47 It comprises, consists essentially of, or consists of an amino acid sequence. In some embodiments, the nucleic acid sequence encoding the SBP transposase polypeptide fused to the NLS is at least 65%, 70%, 75%, 80%, 85%, comprising, consisting essentially of, or consisting of nucleic acid sequences that are 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical can.

In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding an SBP transposase polypeptide fused to an NLS, wherein the SBP transposase polypeptide fused to the NLS is SEQ ID NO: is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to 49 It comprises, consists essentially of, or consists of an amino acid sequence. In some embodiments, the nucleic acid sequence encoding the SBP transposase polypeptide fused to the NLS is at least 65%, 70%, 75%, 80%, 85% any of the sequences set forth in SEQ ID NO:50 comprising, consisting essentially of, or consisting of nucleic acid sequences that are 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to can be done.

In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Sleeping Beauty transposase polypeptide (eg, disclosed in US Pat. No. 9,228,180). In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding an overactive Sleeping Beauty (SB100X) transposase polypeptide. In some embodiments, the Sleeping Beauty transposase is SEQ ID NOs: 51 and 52 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, It comprises or consists of amino acid sequences that are 98%, 99%, or 100% (or any percentage therebetween) identical. In preferred embodiments, the overactive Sleeping Beauty (SB100X) transposase is SEQ ID NOs: 53 and 54 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 comprising, consisting essentially of, or consisting of amino acid sequences that are %, 98%, 99%, or 100% (or any percentage therebetween) identical.

In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a helitron transposase polypeptide (eg, disclosed in WO2019/173636). In some embodiments, the helitron transposase polypeptide is SEQ ID NO: 55 or 56 and at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% comprises, consists essentially of, or consists of amino acid sequences that are %, 99%, or 100% (or any percentage therebetween) identical.

In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Tol2 transposase polypeptide (eg, disclosed in WO2019/173636). In some embodiments, the Tol2 transposase polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% SEQ ID NO: 57 or 58 , 99%, or 100% (or any percentage therebetween) comprise, consist essentially of, or consist of amino acid sequences that are identical.

In some embodiments, the transposase sequence is a TcBuster transposase polypeptide (e.g., disclosed in WO2019/173636) or a mutant TcBuster transposase polypeptide (disclosed in PCT Publication WO2019/173636 and PCT/ described in more detail in US2019/049816). In some embodiments, the TcBuster transposase polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% SEQ ID NO: 59 or 60 , 99%, or 100% (or any percentage therebetween) comprise, consist essentially of, or consist of amino acid sequences that are identical. A polynucleotide encoding a TcBuster transposase can comprise or consist of naturally occurring or non-naturally occurring nucleic acid sequences.

Nanoplasmids for testing liver-specific promoters

The present disclosure provides compositions comprising nanoplasmids for testing liver-specific promoters, referred to herein as "liver nanoplasmids."

In some aspects, a liver nanoplasmid can include at least one piggyBacITR sequence. In some embodiments, a liver nanoplasmid can include at least one insulator sequence. In some embodiments, a liver nanoplasmid can include at least one promoter sequence. In some embodiments, a liver nanoplasmid can include at least one fluorescent protein sequence. In some embodiments, a liver nanoplasmid can include at least one self-cleaving peptide sequence. In some embodiments, a liver nanoplasmid can include at least one luciferase sequence. In some embodiments, a liver nanoplasmid can include at least one poly A sequence.

The liver nanoplasmid comprises a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a fluorescent protein sequence, at least one self-cleaving peptide sequence, a luciferase sequence, a poly A sequence, a second insulator sequence, and a second piggyBacITR sequence. In some embodiments, the liver nanoplasmid comprises, from 5′ to 3′, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a fluorescent protein sequence, at least one self-cleaving peptide sequence, A luciferase sequence, a polyA sequence, a second insulator sequence, and a second piggyBacITR sequence can be included.

In some aspects of the present disclosure, transgene sequences can include fluorescent protein sequences.

In some embodiments, a fluorescent protein sequence can comprise a nucleic acid sequence encoding an eGFP polypeptide. In some embodiments, the fluorescent protein sequence can comprise a nucleic acid sequence encoding an eGFP polypeptide, wherein the eGFP polypeptide is at least 65%, 70%, 75%, 80% SEQ ID NO: 61 or 62 , 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) of amino acid sequences that are identical, or consists of In some embodiments, the nucleic acid sequence encoding the eGFP polypeptide is at least 75%, 80%, 85%, 90%, 95%, 96% any of the sequences set forth in SEQ ID NO: 63, 64, or 133 , 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprise, consist essentially of, or consist of nucleic acid sequences that are 100% identical.

In some aspects of the present disclosure, the transgene sequences can include luciferase sequences.

In some aspects, a luciferase sequence can comprise a nucleic acid sequence encoding a fLuc2 polypeptide. In some embodiments, the luciferase sequence can comprise a nucleic acid sequence encoding a fLuc2 polypeptide, wherein the fLuc2 polypeptide is at least 65%, 70%, 75%, 80%, SEQ ID NO: 65 or 66, comprising, consisting essentially of, or consisting of amino acid sequences that are 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical Become. In some embodiments, the nucleic acid sequence encoding the eGFP polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95% any of the sequences set forth in SEQ ID NO: 67 or 68 , 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical nucleic acid sequences.

In some embodiments, a luciferase sequence can comprise a nucleic acid sequence encoding a nanoluciferase (nLuc) polypeptide. In some embodiments, the nucleic acid sequence encoding the nLuc polypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% any of the sequences set forth in SEQ ID NO: 134. %, 97%, 98%, 99%, or 100% (or any percentage therebetween) that comprise, consist essentially of, or consist of nucleic acid sequences that are identical.

Vector of the present disclosure

The disclosure provides compositions comprising vectors, wherein the vectors comprise at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide. A vector containing at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide is referred to herein as an "AAV piggyBac transposon vector."

The disclosure provides compositions comprising vectors, wherein the vectors comprise at least one AAV transposase polynucleotide. A vector containing at least one AAV transposase polynucleotide is referred to herein as an "AAV transposase vector."

The vectors of this disclosure can be viral vectors or recombinant vectors. Viral vectors can comprise sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors can include sequences isolated or derived from adeno-associated virus (AAV). Viral vectors can include recombinant AAV (rAAV).

Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 , and AAV11). Exemplary adeno-associated and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV), and AAV hybrids containing a genome of one serotype and a capsid of another serotype. Included (eg, AAV2/5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (also called AAV-KP1; Kerun et al. JCI Insight, 2019; 4 (22):el31610) and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303).

The present disclosure provides compositions comprising a plurality of AAV-KP-1 particles comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide. The disclosure provides compositions comprising a plurality of AAV-KP-1 particles comprising at least one AAV transposase polynucleotide. The present disclosure provides a plurality of AAV-KP-1 particles comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide and a plurality of AAV-KP-1 particles comprising at least one AAV transposase polynucleotide. A composition comprising:

The disclosure provides compositions comprising a plurality of AAV-NP59 particles comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide. The disclosure provides compositions comprising a plurality of AAV-NP59 particles comprising at least one AAV transposase polynucleotide. The present disclosure provides compositions comprising a plurality of AAV-NP59 particles comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide and a plurality of AAV-NP59 particles comprising at least one AAV transposase polynucleotide. offer.

Viral vectors and viral particles of the disclosure can be produced using standard methods known in the art.

In some aspects, the AAV-KP-1 particles of the present disclosure can be produced using a KP-1 capsid vector, wherein the KP-1 capsid vector comprises the nucleic acids of SEQ ID NO:70 and SEQ ID NO:71 at least one of the sequences. In some aspects, the AAV-KP-1 particles of the disclosure can be produced using an AAV vector packaging plasmid, wherein the AAV vector packaging plasmid comprises the nucleic acids of SEQ ID NO:75 and SEQ ID NO:76 at least one of the sequences.

In some aspects, the AAV-NP59 particles of the present disclosure can be produced using NP-59 capsid vectors, wherein the NP-59 capsid vectors are SEQ ID NO: 72, SEQ ID NO: 72, SEQ ID NO: 73 , and at least one of the nucleic acid sequences of SEQ ID NO:74. In some aspects, the AAV-NP59 particles of the present disclosure can be produced using an AAV vector packaging plasmid, wherein the AAV vector packaging plasmid comprises the nucleic acid sequences of SEQ ID NO:75 and SEQ ID NO:76. At least one.

A cell delivery composition (eg, polynucleotide, vector) disclosed herein can include a nucleic acid encoding a therapeutic protein or therapeutic agent. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. WO2019/173636 and PCT/US2019/049816. A therapeutic protein can also include, but is not limited to, any of the polypeptides described herein as part of a transgene sequence (eg, OTC, MUT1, etc.).

Formulation, Dosage and Mode of Administration

The disclosure provides formulations, dosages, and methods of administration of the compositions described herein.

The disclosed compositions and pharmaceutical compositions may contain any suitable adjuvant such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants and the like. It can further comprise at least one of the substances. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples of such sterile solutions, and methods for their preparation, include, but are not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. and "Physician's Desk Reference", 52nd ed., Medical Economics (Montvale, NJ) 1998.
Pharmaceutically acceptable agents suitable for the mode of administration, solubility, and/or stability of protein scaffold, fragment or variant compositions, as are well known in the art or described herein. The carrier obtained can be routinely selected.

Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, and may be present alone or in combination, by weight or volume; 1 to 99.99%. Non-limiting examples of protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/protein components that also function in buffer capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides, Examples include raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol, and the like. Preferably, the carbohydrate excipient is mannitol, trehalose and/or raffinose.

The composition may also contain a buffer or pH adjuster, typically a buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. be Preferred buffers are organic acid salts such as citrate.

Additionally, the disclosed compositions may contain polymeric excipients/additives such as polyvinylpyrrolidone, ficoll (high molecular weight sugar), dextrates (eg, cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol. , flavoring agents, antibacterial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g. polysorbates such as "Tween 20" and "Tween 80"), lipids (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol ), and a chelating agent (eg, EDTA).

A number of known and developed modes can be used to administer therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolonic. , intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural , intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrapleural, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous , transdermal, or intravaginal means.

The compositions of the present disclosure are for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly for use in the form of solutions or suspensions; but for use in vaginal or rectal administration in semi-solid forms such as creams and suppositories; for use in buccal or sublingual administration, particularly but not exclusively in tablet or capsule form; or for intranasal use in the form of, but not limited to, powders, drops, aerosols, or certain medicaments; or transdermally, such as, but not limited to, Gels, ointments, lotions, suspensions, or can be prepared for use in transdermal patch systems, which modify skin structure or increase drug concentration in the transdermal patch. (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994), or proteins and peptides with an oxidizing agent to allow application to the skin (WO 98/53847) of a formulation containing a With the application of an electric field, such as foresis, or with the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated by reference in their entirety). are incorporated herein).

For parenteral administration, any of the compositions disclosed herein can be administered as a solution, suspension, emulsion, particle, powder, or lyophilized powder, together with a pharmaceutically acceptable parenteral vehicle. It can be formulated or provided separately. Formulations for parenteral administration can include, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Injectable aqueous or oily suspensions may be prepared according to known methods using suitable emulsifying or wetting agents and suspending agents. Pharmaceutical agents for injection may be non-toxic parenterally administrable diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Water, Ringer's solution, isotonic saline and the like are acceptable vehicles or solvents that can be used. Sterile, fixed oils can be used as a common solvent or suspending medium. For these purposes any type of non-volatile oil and fatty acid can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic mono- or di- or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas pressurized needle-free injection as described in US Pat. No. 5,851,198. devices, and laser drilling devices such as those described in US Pat. No. 5,839,446.

Formulations for oral administration may contain adjuvants such as resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether to artificially increase the permeability of the intestinal wall. and co-administration of enzyme inhibitors (eg, pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. Formulations for the delivery of hydrophilic drugs, including proteins and protein scaffolds, and combinations of at least two surfactants, for oral, buccal, mucosal, nasal, pulmonary, vaginal transmembrane, or rectal administration, are available from US It is described in Patent No. 6,309,663. The active ingredient compound in solid dosage form for oral administration contains at least one excipient (sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin , gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, glycerides). These dosage forms may also contain other types of additives such as inert diluents, lubricants such as magnesium stearate, parabens, preservatives such as sorbic acid, ascorbic acid, α-tocopherol, antioxidants, For example, cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorants, fragrances and the like may be included.

Tablets and pills can be further processed into enteric coated preparations. Preparations for oral administration include emulsions, syrups, elixirs, suspensions and solutions and can be used for medical purposes. These preparations may contain inert diluents commonly used in the field, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (US Pat. No. 4,239,754). Recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used for drug delivery (US Pat. No. 4,925,673). Further, carrier compounds used for oral delivery of bioactive agents are also known in the art, as described in US Pat. Nos. 5,879,681 and 5,871,753. there is

For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower airways or sinuses of the lung. These compositions or pharmaceutical compositions can be delivered by any of the various inhalation or nasal devices known in the art for administration of therapeutic agents by inhalation. These devices capable of depositing an aerosolized formulation in the patient's sinuses or alveoli include metered dose inhalers, nebulizers (eg, jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, and the like. All such devices can employ formulations suitable for administration for aerosol dispensing of the compositions or pharmaceutical compositions described herein. Such aerosols can consist of either solutions (both aqueous and non-aqueous) or solid particles. Additionally, a spray comprising a composition or pharmaceutical composition described herein can be produced by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered dose inhaler (MDI), a propellant, a composition or pharmaceutical composition described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve expels the mixture as an aerosol containing particles preferably in the size range of less than about 10 μm, preferably from about 1 μm to about 5 μm, most preferably from about 2 μm to about 3 μm. A more detailed description of pulmonary administration, formulations, and related devices is disclosed in PCT Publication No. WO2019/049816.

For absorption across mucosal surfaces, the composition comprises an aqueous sequence that facilitates absorption across mucosal surfaces by achieving mucoadhesion of multiple submicron particles, mucoadhesive polymers, bioactive peptides, and emulsion particles. including emulsions containing phases (US Pat. No. 5,514,670). Mucosal surfaces suitable for application of emulsions of the present disclosure can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, can contain as excipients, for example, polyalkylene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal administration may be solid and may contain, for example, lactose as an excipient, or may be an aqueous or oily solution of nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (US Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO2019/049816.

For transdermal administration, the compositions or pharmaceutical compositions disclosed herein may be in the form of liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Enclosed in a delivery device. Synthetic polymers such as polyhydroxy acids, polylactic acids, polyglycolic acid and their copolymers, polyorthoesters, polyanhydrides and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginic acid. Many suitable devices are known containing microparticles made of salts and other polysaccharides, and combinations thereof (US Pat. No. 5,814,599). A more detailed description of transdermal administration, formulations and suitable devices is disclosed in PCT Publication No. WO2019/049816.

It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, eg, from a single dose to a week to a year. Various sustained release, depot, or implant dosage forms are available. For example, dosage forms can include pharmaceutically acceptable non-toxic salts of compounds with low solubility in body fluids: e.g., (a) polybasic acids such as phosphoric acid, sulfuric acid, citric acid, tartaric acid; (b) polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper; salts with cobalt, nickel, cadmium, etc. or with organic cations formed from e.g. N,N'-dibenzylethylenediamine or ethylenediamine, or combinations of (c) (a) and (b), e.g. salt. Additionally, the disclosed compounds, or preferably relatively insoluble salts such as those just mentioned, can be formulated into gels suitable for injection, such as aluminum monostearate gels containing sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoates, and the like. Another type of sustained release depot formulation for injection is a slowly degrading, non-toxic, non-toxic polymer such as polylactic/polyglycolic acid polymers as described, for example, in U.S. Pat. No. 3,773,919. It will contain the dispersed compound or salt for encapsulation in the antigenic polymer. Compounds, or preferably relatively insoluble salts such as those described above, can also be formulated into cholesterol matrix silastic pellets, particularly for use in animals. Additional sustained release, depot, or implant formulations, such as gas or liquid liposomes, are known in the literature (US Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed. , Marcel Dekker, Inc., N.Y., 1978).

Appropriate dosages are well known in the art. For example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000) Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, N.J. sea bream. Preferred dosages include serum concentrations of about 0.1-5000 μg/ml per single or multiple doses, or optionally about 0.1 μg/ml to achieve any range, value, or fraction thereof. ~99 and/or 100-500 mg/kg/dose, or any range, value or fraction thereof. A preferred dosage range of a composition or pharmaceutical composition disclosed herein is about 1 mg, up to about 3 mg, about 6, or about 12 mg per kg body weight of a subject.

Alternatively, the dose administered will depend on known factors, such as the pharmacodynamic properties of the particular drug and its mode and route of administration, the age, health, and weight of the recipient. It may vary depending on the nature and extent of symptoms, type of concomitant therapy, frequency of treatment, desired effect, and the like. Generally, the dosage of active ingredient can be about 0.1-100 milligrams per kilogram of body weight. A dose of 0.1 to 50 mg, preferably 0.1 to 10 mg/kg, in single dose or sustained release form is usually effective to obtain the desired results.

As a non-limiting example, for treatment of humans or animals, as a single or periodic dose of the compositions or pharmaceutical compositions disclosed herein, at least one day for 1 to 40 days, or alternatively or additionally for at least 1 week from 1 to 52 weeks, or alternatively or additionally for at least 1 year from 1 to 20 years, or any combination thereof using single, infusion, or multiple doses. can be provided from about 0.1 to 100 mg/kg per day, or any range, value, or fraction thereof.

Dosage forms suitable for internal administration generally contain from about 0.001 mg to about 500 mg of active ingredient per unit or container. In these pharmaceutical compositions the active ingredient is usually present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.

Effective amounts are from about 0.1 to 5000 μg/ml per single or multiple doses, as determined using known methods described herein or known in the relevant arts. Amounts of about 0.001 to about 500 mg/kg can be included per single (eg, bolus), multiple, or continuous administration to achieve serum concentrations, or any range, value, or portion thereof. can.

In embodiments where the composition administered to a subject in need thereof is the modified cells disclosed herein, the cells are from about 1×10 ³ to 1×10 ¹⁵ cells, from about 1×10 ⁴ to 1×10 ^{12 cells.} cells, about 1×10 ⁵ to 1×10 ¹⁰ cells, about 1×10 ⁶ to 1×10 ⁹ cells, about 1×10 ⁶ to 1×10 ⁸ cells, about 1×10 ⁶ to 1×10 ⁷ cells, or about 1×10 ⁶ to 25×10 ⁶ cells. In one embodiment, cells are administered at about 5×10 ⁶ to 25×10 ⁶ cells.

More detailed descriptions of pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions are disclosed in PCT Publication No. WO2019/049816.

Methods of Using the Compositions of the Disclosure

The present disclosure uses the disclosed compositions or pharmaceutical compositions to administer or contact, for example, a cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. Accordingly, any disclosed composition or pharmaceutical composition for treating a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or described herein Provide use of things. In one embodiment the subject is a mammal. Preferably the subject is human. The terms "subject" and "patient" are used interchangeably herein.

The present disclosure includes administering to a subject in need thereof at least one therapeutically effective amount of at least one composition of the present disclosure to treat at least one metabolic liver disorder (MLD) in said subject. Provide a method for treatment.

The present disclosure provides at least one composition of the present disclosure for use in treating at least one metabolic liver disorder in a subject, wherein said at least one composition comprises at least one therapeutically effective amount of for administration to a subject at

The disclosure provides use of at least one composition of the disclosure for the manufacture of a medicament for treating at least one metabolic liver disorder in a subject, wherein the at least one composition comprises for administration to a subject in at least one therapeutically effective amount.

In some aspects of the foregoing methods and uses, at least one composition of the disclosure can comprise at least one AAVpiggyBac transposon vector of the disclosure.

Accordingly, the present disclosure treats at least one metabolic liver disorder in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of at least one of at least one AAVpiggyBac transposon vector of the present disclosure. provide a method for

The present disclosure provides at least one AAVpiggyBac transposon vector of the present disclosure for use in treating at least one metabolic liver disorder in a subject, wherein said at least one AAVpiggyBac transposon vector comprises at least one treatment It is for administration to a subject in an effective amount.

The disclosure provides use of at least one AAVpiggyBac transposon vector of the disclosure for the manufacture of a medicament for the treatment of at least one metabolic liver disorder in a subject, wherein said at least one AAVpiggyBac transposon vector is for administration to a subject in at least one therapeutically effective amount.

In some aspects of the aforementioned methods and uses, at least one composition of the disclosure can comprise at least one AAV transposase vector of the disclosure.

Accordingly, the present disclosure provides for the treatment of at least one metabolic liver disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of at least one of at least one AAV transposase vector of the present disclosure. Provide a method for treatment.

The disclosure provides at least one AAV transposase vector of the disclosure for use in treating at least one metabolic liver disorder in a subject, wherein said at least one AAV transposase vector comprises for administration to a subject in at least one therapeutically effective amount.

The disclosure provides use of at least one AAV transposase vector of the disclosure for the manufacture of a medicament for treating at least one metabolic liver disorder in a subject, wherein said at least one AAV The transposase vector is for administration to a subject in at least one therapeutically effective amount.

The present disclosure provides a method of treating at least one metabolic liver disorder in a subject, comprising administering to the subject: a) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a transposon; b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase, wherein said transposon is at least one therapeutic protein comprising a nucleotide sequence that encodes

In some aspects of the aforementioned methods, the composition containing the transposon-containing nucleic acid molecule can be any AAV piggyBac transposon vector described herein.

In some aspects of the foregoing methods, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be any AAV transposase vector of the present disclosure.

Accordingly, the present disclosure provides a method of treating at least one metabolic liver disorder in a subject, comprising administering to the subject: a) a therapeutically effective amount of at least one of at least one AAVpiggyBac transposon vector of the present disclosure; and b) a therapeutically effective amount of at least one of at least one AAV transposase vector of the present disclosure.

Accordingly, the disclosure provides a combination of at least one AAV piggyBac transposon vector of the disclosure and at least one AAV transposase vector of the disclosure for use in treating at least one metabolic liver disorder in a subject. wherein said at least one AAV piggyBac transposon vector is for administration to a subject in at least one therapeutically effective amount, and said at least one AAV transposase vector is in at least one therapeutically effective amount for administration to a subject.

Accordingly, the present disclosure provides use of a combination of at least one AAV piggyBac transposon vector of the present disclosure and at least one AAV transposase vector of the present disclosure in the manufacture of a medicament for the treatment of at least one metabolic disease in a subject. wherein said at least one AAV piggyBac transposon vector is for administration to a subject in at least one therapeutically effective amount, and said at least one AAV transposase vector is for at least one therapeutic It is for administration to a subject in an effective amount.

Metabolic liver disorders include, but are not limited to, urea cycle disorders, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperarginine blood), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof. In some aspects, the metabolic liver disorder is ornithine transcarbamylase (OTC) deficiency.

In some embodiments of the foregoing methods, a composition comprising a nucleic acid molecule comprising a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered simultaneously, wherein said transposon comprises a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, a composition comprising a nucleic acid molecule comprising a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered sequentially, wherein Said transposon comprises a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, a composition comprising a nucleic acid molecule comprising a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered in close temporal proximity. , wherein said transposon comprises a nucleotide sequence encoding at least one therapeutic protein.

As used herein, the term "temporal proximity" means that administration of one therapeutic composition (eg, a composition comprising a transposon) is closely related to administration of another therapeutic composition (eg, a composition comprising a transposase). Refers to occurring within a period of time before or after administration such that the therapeutic effect of one therapeutic agent overlaps with that of another therapeutic agent. In some embodiments, the therapeutic effect of one therapeutic agent completely overlaps with the therapeutic effect of another therapeutic agent. In some embodiments, "temporal proximity" means that administration of one therapeutic agent occurs within a period of time before or after administration of another therapeutic agent, thus one therapeutic agent and the other therapeutic agent. There is a synergistic effect between "Temporal proximity" includes, but is not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to whom the therapeutic agent is administered; therapeutic outcome to be achieved; dosage, frequency, and duration of administration of the therapeutic; pharmacokinetics and pharmacodynamics of the therapeutic; and the route by which the therapeutic is administered. In some embodiments, "temporal proximity" is within 15 minutes, within 30 minutes, within 1 hour, within 2 hours, within 4 hours, within 6 hours, within 8 hours, within 12 hours, within 18 hours. , within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 1 week, within 2 weeks, within 3 weeks, within 4 weeks, within 6 weeks, 8 means within a week. In some embodiments, multiple administrations of one therapeutic agent can be administered in close temporal proximity to a single administration of another therapeutic agent. In some embodiments, temporal proximity may vary during treatment cycles or with dosing regimens.

In some aspects of the therapeutic methods of the present disclosure, administration of at least one composition and/or vector of the present disclosure to a subject causes exogenous protein (e.g., therapeutic proteins, transposases, etc.).

In some embodiments, administration of at least one composition and/or vector of the disclosure results in at least about 10%, or at least about 15%, or at least about 20% of the cells in the tissue and/or organ, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the exogenous protein bring about expression.

In some embodiments, administration of at least one composition and/or vector of the present disclosure results in at least about 10%, or at least about 15%, of a specific subset or subsets of cells in a tissue and/or organ. %, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least In about 99% it results in expression of the exogenous protein.

In some embodiments, administration of at least one composition and/or vector of the present disclosure is administered to a tissue and/or organ for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days. days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

In some embodiments, administration of at least one composition and/or vector of the present disclosure affects a specific subset or subsets of cells in a tissue and/or organ for at least about 1 day, or for at least about 2 days. days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days , resulting in expression of the exogenous protein.

In some embodiments, administration of at least one composition and/or vector of the present disclosure takes about 1 day or less, or about 2 days or less, or about 3 days or less, or about 4 days or less in a tissue and/or organ. days or less, or about 5 days or less, or about 6 days or less, or about 7 days or less, or about 8 days or less, or about 9 days or less, or about 10 days or less.

In some embodiments, administration of at least one composition and/or vector of the present disclosure affects a specific subset or subsets of cells within a tissue and/or organ for no more than about 1 day, or for about 2 days. days or less, or about 3 days or less, or about 4 days or less, or about 5 days or less, or about 6 days or less, or about 7 days or less, or about 8 days or less, or about 9 days or less, or about 10 days or less of the exogenous protein.

In some embodiments, the tissue and/or organ can be liver. In some embodiments, the particular one or more subsets of cells include, but are not limited to, hepatocytes, hepatic stellate cells, Kupffer cells, hepatic sinusoidal endothelial cells, or any combination thereof. can contain.

Any method of the present disclosure includes administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal, or animal in need of such modulation, treatment, or therapy. can include administering to a subject. Such methods can optionally further comprise co-administration or combination therapy to treat such diseases or disorders, wherein any composition or medicament disclosed herein Administering the composition further comprises concurrently and/or subsequently administering at least one additional treatment for a urea cycle disorder.

Further treatments for urea cycle disorders include, but are not limited to, dialysis, hemofiltration, calorie replacement, hormonal suppression, glucose infusion, insulin infusion, pharmacological removal of excess nitrogen, administration of dextrose, administration of fluids. , administration of Intralipid®, administration of ammonia scavenger, administration of arginine, administration of sodium phenylacetate, administration of sodium benzoate, administration of Ammonul, administration of phenylbutyric acid, citrulline supplementation, arginine supplementation, or any of these Combinations may be included.

Exemplary Implementation of the Disclosure

Embodiment 1. An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising in the 5' to 3' direction:
a) a first AAV inverted terminal repeat (ITR) sequence,
b) a first piggyBacITR sequence,
c) a first insulator sequence;
d) at least one promoter sequence;
e) at least one transgene sequence,
f) a poly A sequence,
g) a second insulator array;
h) a second piggyBacITR sequence, and i) a second AAVITR sequence.

Embodiment 2. The AAVpiggyBac transposon polynucleotide of embodiment 1, wherein the AAVpiggyBac transposon polynucleotide comprises DNA, cDNA, gDNA, RNA, mRNA, or any combination thereof.

Embodiment 3. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first and/or second AAVITR sequences comprise the nucleic acid sequences of any one of SEQ ID NOs: 1-4, 93-94, 105-106, and 127. .

Embodiment 4. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:3 and the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:4.

Embodiment 5. The AAV piggyBac of any of the preceding embodiments, wherein the first piggyBacITR sequence and/or the second piggyBacITR sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 5-6, 86-90, 95-96, and 125. transposon polynucleotide.

Embodiment 6. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:5 and the second piggyBacITR comprises the nucleic acid of SEQ ID NO:6.

Embodiment 7. AAVpiggyBac according to any of the preceding embodiments, wherein the first insulator sequence and/or the second insulator sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 7-8, 77-80, and 91-92. transposon polynucleotide.

Embodiment 8. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO:7 and the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO:8.

Embodiment 9. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence is a liver-specific promoter.

Embodiment ten. Liver-specific promoters include hybrid liver promoter (HLP), LP1 promoter, leukocyte-specific expression of pp52 (LSP1) long promoter, thyroxine-binding globulin (TBG) promoter, wTBG promoter, liver combinatorial bundle (HCB) promoter, 2xApoE-hAAT The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, which is a promoter or leukocyte-specific expression of the pp52 (LSP1) and chimeric intron promoters.

Embodiment 11. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 9-16, 69, 107, 126, and 132.

Embodiment 12. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence comprises a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide.

Embodiment 13. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the MUT1 polypeptide comprises the amino acid sequence of SEQ ID NOs: 17, 18, 121, or 122.

Embodiment 14. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the nucleic acid sequence encoding the MUT1 polypeptide comprises the nucleic acid sequences of SEQ ID NOs: 19, 20, or 111-120.

Embodiment 15. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence comprises a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide.

Embodiment 16. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the OTC polypeptide comprises the amino acid sequence of SEQ ID NO:21 or 81.

Embodiment 17. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the nucleic acid sequence encoding the OTC polypeptide comprises the nucleic acid sequence of any of SEQ ID NOS:22, 23, 82, and 83.

Embodiment 18. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence comprises a nucleic acid sequence encoding an iCas9 polypeptide.

Embodiment 19. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the iCas9 polypeptide comprises the amino acid sequence of SEQ ID NO:24 or 84.

Embodiment 20. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the nucleic acid sequence encoding the iCas9 polypeptide comprises the nucleic acid sequence of SEQ ID NO:25 or 85.

Embodiment 21. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence is operably linked to at least one promoter sequence.

Embodiment 22. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein expression of at least one transgene sequence is controlled by at least one promoter sequence.

Embodiment 23. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the polyA sequence comprises the nucleic acid sequence of any one of SEQ ID NOS:26-27, 97, 108, 128, and 136.

Embodiment 24. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the AAVpiggyBac transposon polynucleotide further comprises at least a second transgene sequence.

Embodiment 25. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least the second transgene sequence comprises a nucleic acid sequence encoding an iCas9 polypeptide.

Embodiment 26. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the iCas9 polypeptide comprises the amino acid sequence of SEQ ID NO:24 or 84.

Embodiment 27. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the nucleic acid sequence encoding the iCas9 polypeptide comprises the nucleic acid sequence of SEQ ID NO:25 or 85.

Embodiment 28. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least the second transgene sequence comprises a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide.

Embodiment 29. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the MUT1 polypeptide comprises the amino acid sequence of SEQ ID NOs: 17, 18, 121, or 122.

Embodiment 30. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the nucleic acid sequence encoding the MUT1 polypeptide comprises the nucleic acid sequences of SEQ ID NOs: 19, 20, or 111-120.

Embodiment 31. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least the second transgene sequence comprises a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide.

Embodiment 32. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the OTC polypeptide comprises the amino acid sequence of SEQ ID NO:21 or 81.

Embodiment 33. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the nucleic acid sequence encoding the OTC polypeptide comprises the nucleic acid sequence of any of SEQ ID NOS:22, 23, 82, and 83.

Embodiment 34. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the AAVpiggyBac transposon polynucleotide further comprises at least a second promoter sequence.

Embodiment 35. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least a second promoter sequence is located between the at least one transgene sequence and the at least a second transgene sequence.

Embodiment 36. The AAVpiggyBac transposon polynucleotide further comprises at least one self-cleaving peptide sequence, wherein the at least one self-cleaving peptide sequence is T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, which is a peptide, or a nucleic acid sequence encoding a GSG-P2A peptide.

Embodiment 37. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one self-cleaving peptide sequence is located between at least one transgene sequence and at least a second transgene sequence.

Embodiment 38. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the AAVpiggyBac transposon polynucleotide comprises at least two transgene sequences.

Embodiment 39. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the at least two transgene sequences are the same sequence.

Embodiment 40. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the at least two transgene sequences are different sequences.

Embodiment 41. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, further comprising at least one DNA spacer sequence.

Embodiment 42. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 103, 109, 129-131, and 137.

Embodiment 43. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the AAVpiggyBac transposon polynucleotide comprises at least two promoter sequences.

Embodiment 44. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the at least two promoter sequences are the same sequence.

Embodiment 45. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the at least two promoter sequences are different sequences.

Embodiment 46A. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence,
b) a first piggyBacITR sequence,
c) a first insulator sequence;
d) at least one promoter sequence;
e) at least one transgene sequence,
f) a poly A sequence,
g) a second insulator array;
h) a second piggyBacITR sequence;
i) at least one DNA spacer sequence, and j) a second AAVITR sequence.

Embodiment 46B. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:3.

Embodiment 46C. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 46D. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 46E. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO:7.

Embodiment 46F. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:9.

Embodiment 46G. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:126.

Embodiment 46H. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence comprises the nucleic acid sequence of SEQ ID NO:22.

Embodiment 46I. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the poly A sequence comprises the nucleic acid sequence of SEQ ID NO:97.

Embodiment 46J. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO:8.

Embodiment 46K. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:96.

Embodiment 46L. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO:129.

Embodiment 46M. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:4.

Embodiment 46N. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:95 or SEQ ID NO:125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:126;
e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96;
i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:129; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 47. The AAVpiggyBac transposon polynucleotide of any one of embodiments 46A-46N, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:9.

Embodiment 48. The AAVpiggyBac transposon polynucleotide of any one of embodiments 46A-46N, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:126.

Embodiment 49. The AAGpiggyBac transposon polynucleotide of any one of embodiments 46A-46N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 50. The AAGpiggyBac transposon polynucleotide of any one of embodiments 46A-46N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 51. The AAVpiggyBac transposon polynucleotide of any one of embodiments 46A-50, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:138.

Embodiment 52A. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence,
b) a first piggyBacITR sequence,
c) a first insulator sequence;
d) at least one promoter sequence;
e) at least one transgene sequence,
f) a poly A sequence,
g) a second insulator array;
h) a second piggyBacITR sequence,
i) at least one DNA spacer sequence, and j) a second AAVITR sequence.

Embodiment 52B. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:3.

Embodiment 52C. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 52D. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 52E. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO:7.

Embodiment 52F. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:10.

Embodiment 52G. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:132.

Embodiment 52H. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence comprises the nucleic acid sequence of SEQ ID NO:22.

Embodiment 52I. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the poly A sequence comprises the nucleic acid sequence of SEQ ID NO:97.

Embodiment 52J. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO:8.

Embodiment 52K. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:96.

Embodiment 52L. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO:130.

Embodiment 52M. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:4.

Embodiment 52N. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:95 or SEQ ID NO:125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 10 or SEQ ID NO: 132;
e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96;
i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:130; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 53. The AAVpiggyBac transposon polynucleotide of any one of embodiments 52A-52N, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:10.

Embodiment 54. The AAVpiggyBac transposon polynucleotide of any one of embodiments 52A-52N, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:132.

Embodiment 55. The AAGpiggyBac transposon polynucleotide of any one of embodiments 52A-52N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 56. The AAGpiggyBac transposon polynucleotide of any one of embodiments 52A-52N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 57. The AAVpiggyBac transposon polynucleotide of any one of embodiments 52A-56, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:139.

Embodiment 58A. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence,
b) a first piggyBacITR sequence,
c) a first insulator sequence;
d) at least one promoter sequence;
e) at least one transgene sequence,
f) a poly A sequence,
g) a second insulator array;
h) a second piggyBacITR sequence;
i) at least one DNA spacer sequence, and j) a second AAVITR sequence.

Embodiment 58B. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:3.

Embodiment 58C. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 58D. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 58E. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO:7.

Embodiment 58F. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:13.

Embodiment 58G. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transgene sequence comprises the nucleic acid sequence of SEQ ID NO:22.

Embodiment 58H. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the poly A sequence comprises the nucleic acid sequence of SEQ ID NO:97.

Embodiment 58I. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO:8.

Embodiment 58J. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:96.

Embodiment 58K. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO:131.

Embodiment 58L. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:4.

Embodiment 58M. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:95 or SEQ ID NO:125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 13;
e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96;
i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:131; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 59. The AAGpiggyBac transposon polynucleotide of any one of embodiments 58A-58M, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 60. The AAGpiggyBac transposon polynucleotide of any one of embodiments 58A-58M, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 61. The AAVpiggyBac transposon polynucleotide of any one of embodiments 58A-60, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:140.

Embodiment 62. An AAVpiggyBac transposon polynucleotide comprising in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:95 or SEQ ID NO:125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:126;
e) a first transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a first self-cleaving peptide sequence comprising the nucleic acid sequence of SEQ ID NO:31;
g) a second transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 133;
h) at least a second self-cleaving peptide sequence comprising the nucleic acid sequence of SEQ ID NO:32;
i) at least a third transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 134;
j) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
k) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
l) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96; and m) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 63. 63. The AAGpiggyBac transposon polynucleotide of embodiment 62, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 64. 63. The AAGpiggyBac transposon polynucleotide of embodiment 62, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 65. 63. The AAVpiggyBac transposon polynucleotide of embodiment 62, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:9.

Embodiment 66. 63. The AAVpiggyBac transposon polynucleotide of embodiment 62, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:126.

Embodiment 67. 67. The AAVpiggyBac transposon polynucleotide of any one of embodiments 62-66, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:141.

Embodiment 68. An AAVpiggyBac transposon polynucleotide comprising in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:95 or SEQ ID NO:125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 10 or SEQ ID NO: 132;
e) a first transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a first self-cleaving peptide sequence comprising the nucleic acid sequence of SEQ ID NO:31;
g) a second transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 133;
h) at least a second self-cleaving peptide sequence comprising the nucleic acid sequence of SEQ ID NO:32;
i) at least a third transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 134;
j) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
k) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
l) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96; and m) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 69. 69. The AAGpiggyBac transposon polynucleotide of embodiment 68, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 70. 69. The AAGpiggyBac transposon polynucleotide of embodiment 68, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 71. 69. The AAVpiggyBac transposon polynucleotide of embodiment 68, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:10.

Embodiment 72. 69. The AAVpiggyBac transposon polynucleotide of embodiment 68, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:132.

Embodiment 73. 73. The AAVpiggyBac transposon polynucleotide of any one of embodiments 68-72, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:142.

Embodiment 74. An AAVpiggyBac transposon polynucleotide comprising in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:95 or SEQ ID NO:125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 13;
e) a first transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) at least one self-cleaving peptide sequence comprising the nucleic acid sequence of SEQ ID NO: 135;
g) at least a second transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 134;
h) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
i) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
j) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96; and k) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 75. 75. The AAGpiggyBac transposon polynucleotide of embodiment 74, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:95.

Embodiment 76. 75. The AAGpiggyBac transposon polynucleotide of embodiment 74, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO:125.

Embodiment 77. 77. The AAVpiggyBac transposon polynucleotide of any one of embodiments 74-76, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:143.

Embodiment 78. A vector comprising the AAVpiggyBac transposon polynucleotide of any of the preceding embodiments.

Embodiment 79. The vector of any of the preceding embodiments, wherein the vector is a viral vector.

Embodiment 80. The vector of any of the preceding embodiments, wherein the viral vector is an adeno-associated virus (AAV) viral vector.

Embodiment 81. The vector of any of the preceding embodiments, wherein the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector.

Embodiment 82. The vector of any of the preceding embodiments, wherein the AAV viral vector is an AAV-KP-1 or AAV-NP59 viral vector, preferably the AAV viral vector is an AAV-KP-1 viral vector.

Embodiment 83. A composition comprising the vector of any of embodiments 78-82.

Embodiment 84. An AAV transposase polynucleotide comprising, from 5' to 3', a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a poly A sequence, and a second AAVITR sequence. .

Embodiment 85. 85. The AAV transposase polynucleotide of embodiment 84, wherein the AAVpiggyBac transposon polynucleotide comprises DNA, cDNA, gDNA, RNA, mRNA, or any combination thereof.

Embodiment 86. The AAV transposase polypeptide of any of the preceding embodiments, wherein the first and/or second AAVITR sequences comprise the nucleic acid sequences of any of SEQ ID NOs: 1-4, 93-94, 105-106, and 127. nucleotide.

Embodiment 87. The AAV transposase polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:1 and the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:2.

Embodiment 88. The AAV transposase polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:105 and the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:106.

Embodiment 89. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence is a liver-specific promoter.

Embodiment 90. Liver-specific promoters include hybrid liver promoter (HLP), LP1 promoter, leukocyte-specific expression of pp52 (LSP1) long promoter, thyroxine-binding globulin (TBG) promoter, wTBG promoter, liver combinatorial bundle (HCB) promoter, 2xApoE-hAAT The AAV transposase polynucleotide of any of the preceding embodiments, which is a promoter or leukocyte-specific expression of the pp52 (LSP1) and chimeric intron promoters.

Embodiment 91. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of any of SEQ ID NOs: 9-16, 69, 107, 126, and 132.

Embodiment 92. A nucleic acid in which at least one transposase sequence encodes a piggyBac™ (PB) transposase polypeptide, a piggyBac-like (PBL) transposase polypeptide, or a SuperpiggyBac™ (SPB) transposase polypeptide The AAV transposase polynucleotide of any of the preceding embodiments, comprising the sequence.

Embodiment 93. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one transposase sequence comprises a nucleic acid sequence encoding the amino acid sequence of any of SEQ ID NOS:39-42, 47, and 49.

Embodiment 94. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one transposase sequence comprises the nucleic acid sequence of SEQ ID NO:48 or 50.

Embodiment 95. at least one transposase sequence is a sleeping beauty transposase polypeptide, a hyperactive sleeping beauty (SB100X) transposase polypeptide, a helitron transposase polypeptide, a Tol2 transposase polypeptide, TcBuster The AAV transposase polynucleotide of any of the preceding embodiments, comprising a nucleic acid sequence encoding a transposase polypeptide or a mutated TcBuster transposase polypeptide.

Embodiment 96. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one transposase sequence comprises a nucleic acid sequence encoding the amino acid sequence of any of SEQ ID NOs:51-60.

Embodiment 97. The AAV transposase polynucleotide of any of the preceding embodiments, wherein the poly A sequence comprises the nucleic acid sequence of SEQ ID NOs:26-27, 97, or 108.

Embodiment 98. The AAVpiggyBac transposon polynucleotide of any of the preceding embodiments, wherein at least one transposase sequence is operably linked to at least one promoter sequence.

Embodiment 99. The AAV piggyBac transposon polynucleotide of any of the preceding embodiments, wherein expression of at least one transposase sequence is controlled by at least one promoter sequence.

Embodiment 100. The AAV transposase polynucleotide of any of the preceding embodiments, wherein the AAV transposase polynucleotide further comprises at least one DNA spacer sequence.

Embodiment 101. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 103 or 109.

Embodiment 102A. An AAV transposase polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence,
b) at least one promoter sequence at least one promoter sequence;
c) at least one transposase sequence;
d) a poly A sequence,
e) at least one DNA spacer sequence, and f) a second AAVITR sequence.

Embodiment 102B. The AAV transposase polynucleotide of any of the preceding embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO:127.

Embodiment 102C. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:9.

Embodiment 102D. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:126.

Embodiment 102E. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one transposase sequence comprises the nucleic acid sequence of SEQ ID NO:48.

Embodiment 102F. The AAV transposase polynucleotide of any of the preceding embodiments, wherein the poly A sequence comprises the nucleic acid sequence of SEQ ID NO:136.

Embodiment 102G. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO:137.

Embodiment 102H. The AAV transposase polynucleotide of any of the preceding embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO:4.

Embodiment 102I. An AAV transposase polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 127;
b) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:126;
c) at least one transposase sequence comprising the nucleic acid sequence of SEQ ID NO:48;
d) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO: 136;
e) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:137; and f) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4.

Embodiment 103. The AAV transposase polynucleotide of any one of embodiments 102A-102I, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:9.

Embodiment 104. The AAV transposase polynucleotide of any one of embodiments 102A-102I, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:126.

Embodiment 105. The AAVpiggyBac transposase polynucleotide of embodiments 102A-104, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO:144.

Embodiment 106. A vector comprising the AAV transposase polynucleotide of any of the preceding embodiments.

Embodiment 107. The vector of any of the preceding embodiments, wherein the vector is a viral vector.

Embodiment 108. The vector of any of the preceding embodiments, wherein the viral vector is an adeno-associated virus (AAV) viral vector.

Embodiment 109. The vector of any of the preceding embodiments, wherein the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector.

Embodiment 110. The vector of any of the preceding embodiments, wherein the AAV viral vector is an AAV-KP-1 or AAV-NP59 AAV viral vector, preferably the AAV viral vector is an AAV-KP-1 viral vector.

Embodiment 111. A composition comprising the vector of any of embodiments 102-110.

Embodiment 112. A composition comprising the vector of any of embodiments 78-82 and the vector of any of embodiments 102-110.

Embodiment 113. A method of treating at least one metabolic liver disorder (MLD) in a subject in need thereof, comprising at least one treatment of at least one polynucleotide, vector, or composition of any of the preceding embodiments A method comprising administering an effective amount to a subject.

Embodiment 114. A method of treating at least one metabolic liver disorder (MLD) in a subject in need thereof comprising administering to the subject:
a) at least one therapeutically effective amount of the AAVpiggyBac transposon polynucleotide of any one of the preceding embodiments or of any one of the vectors and/or compositions of the preceding embodiments comprising the AAVpiggyBac transposon polynucleotide; and b. ) at least one therapeutically effective amount of the AAVpiggyBac transposase polynucleotide of any one of the preceding embodiments or of any one of the vectors and/or compositions of the preceding embodiments comprising an AAVpiggyBac transposase polynucleotide; .

Embodiment 115. at least one MLD, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) ) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), Methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof.

Embodiment 116. 116. The method of embodiment 115, wherein the MLD is ornithine transcarbamylase (OTC) deficiency.

definition

nucleic acid and polynucleotide molecules

Nucleic acid and polynucleotide molecules of the present disclosure may be in the form of RNA, such as, but not limited to, mRNA, hnRNA, tRNA, or any other form, or clonally or synthetically, or combinations thereof. It can be in the form of DNA, including cDNA and genomic DNA obtained by. The DNA can be triple stranded, double stranded, or single stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

Construction of Nucleic Acid and Polynucleotide Molecules

The nucleic acid and polynucleotide molecules of the present disclosure are produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof known in the art. be able to.

Nucleic acid and polynucleotide molecules can conveniently include nucleotide sequences in addition to the polynucleotides of the present disclosure. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into a nucleic acid to aid in polynucleotide isolation. Also, translatable sequences can be inserted to aid in the isolation of translated polynucleotides of this disclosure. A hexahistidine marker sequence, for example, provides a convenient means of purifying the proteins of the present disclosure. Nucleic acids of the disclosure, excluding coding sequences, are optionally vectors, adapters or linkers for cloning and/or expression of polynucleotides of the disclosure.

Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of polynucleotides, or to facilitate introduction of polynucleotides into cells. can be improved. The use of cloning vectors, expression vectors, adapters and linkers are well known in the art.

Recombinant methods for constructing nucleic acid and polynucleotide molecules

Nucleic acid and polynucleotide molecules of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, may be obtained from biological sources using any number of cloning methods known to those of skill in the art. be able to. In some embodiments, oligonucleotide probes that selectively hybridize to polynucleotides of the present disclosure under stringent conditions are used to identify desired sequences in cDNA or genomic DNA libraries. RNA isolation and cDNA and genomic library construction are well known to those of skill in the art.

Nucleic acid screening and isolation methods

cDNA or genomic libraries can be screened using probes based on the polynucleotide sequences of the present disclosure. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that varying degrees of stringency of hybridization can be used in the assay, and that either the hybridization or the wash medium can be stringent. As hybridization conditions become more stringent, a greater degree of complementarity between probe and target is required for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of partially denaturing solvents such as formamide. For example, the stringency of hybridization is conveniently altered by varying the polarity of the reactant solutions, eg, by manipulating the concentration of formamide within the range of 0%-50%. The degree of complementarity (sequence identity) required for detectable binding will vary according to the stringency of the hybridization medium and/or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therebetween. However, it should be understood that slight sequence variations in probes and primers can be compensated for by reducing the stringency of the hybridization medium and/or wash medium.

Methods for amplifying RNA or DNA are known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limited to, the polymerase chain reaction (PCR) and related amplification processes (e.g. Mullis, et al., US Pat. No. 4,683,195; 4,683,202, 4,800,159, 4,965,188; Tabor et al. 4,795,699 and 4,921,794; Wilson, et al. 5,122,464; Innis 5,091,310; Gyllensten, et al. 5,066,584; Gelfand, et al. 4,889,818; Silver, et al. 4,994,370; Biswas 4,766,067; Ringold 4,656,134), and for double-stranded DNA synthesis. and RNA-mediated amplification (Malek, et al., U.S. Pat. No. 5,130,238, tradename NASBA) using antisense RNA to the target sequence as a template for the full contents of these references. incorporated herein by.

For example, polymerase chain reaction (PCR) technology can be used to amplify sequences of polynucleotides of the present disclosure, and sequences of related genes, directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods are also used, e.g., for cloning nucleic acid sequences that encode proteins to be expressed, for detecting the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other methods. It can be useful for making nucleic acids for use as probes for the purpose. Examples of techniques sufficient to direct one skilled in the art to in vitro amplification methods are U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds. , Academic Press Inc., San Diego, Calif. (1990). Commercial kits for genomic PCR amplification are known in the art. See, eg, Advantage-GC Genomic PCR Kit (Clontech). Additionally, for example, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

Synthetic methods for constructing nucleic acids

Nucleic acid and polynucleotide molecules of the disclosure can also be prepared by direct chemical synthesis by known methods. Chemical synthesis usually produces a single-stranded oligonucleotide, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 bases or more, longer sequences can be obtained by ligation of shorter sequences.

Recombinant expression cassette

The disclosure further provides recombinant expression cassettes comprising nucleic acid or polynucleotide molecules of the disclosure. A recombinant expression cassette can be constructed using the nucleic acids or polynucleotides of the disclosure and introduced into at least one desired host cell. A recombinant expression cassette typically comprises a polynucleotide of this disclosure operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of this disclosure.

In some embodiments, an isolated nucleic acid that functions as a promoter, enhancer, or other element is introduced into a non-heterologous form of a polynucleotide of the disclosure at a suitable position (upstream, downstream, or within an intron). , can upregulate or downregulate the expression of the polynucleotides of the present disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and/or substitution.

Expression vectors and host cells

The present disclosure also relates to vectors containing the isolated nucleic acid and polynucleotide molecules of the present disclosure, host cells genetically engineered with recombinant vectors, and production of at least the polynucleotides by recombinant techniques well known in the art.

The polynucleotide can optionally be ligated into a vector containing a selectable marker for propagation in a host. Plasmid vectors are generally introduced into a precipitate, such as a calcium phosphate precipitate, or complexed with charged lipids. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and transduced into host cells.

A DNA insert should be operably linked to a suitable promoter. Expression constructs further contain sites for transcription initiation, transcription termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably includes a translation initiation codon at the beginning and a stop codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, UAA and UAG are preferred for expression in mammalian or eukaryotic cells.

Expression vectors preferably but optionally include at least one selectable marker. Such markers include, but are not limited to, ampicillin, zeocin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/ geneticin (neo gene), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), mycophenolic acid, or glutamine synthase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359) No. 5,827,739), blasticidin (bsd gene), resistance gene, and ampicillin, zeocin (Shbla gene), puromycin (pac gene) for culturing E. coli and other bacteria or prokaryotes, hygromycin B (hygB gene), G418/geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes (the above patents are incorporated by reference in their entireties). incorporated herein). Appropriate media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods.

The expression vector preferably but optionally contains at least one selectable cell surface marker for isolating cells that have been modified by the compositions and methods of this disclosure. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells that have been modified by the compositions or methods of this disclosure from cells that have not been modified by the compositions or methods of this disclosure. Such cell surface markers include, but are not limited to, "designated cluster" or "classic determinant" proteins (often abbreviated as "CD"), such as truncated or full-length Types CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof are included. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8):1277-87).

The expression vector preferably but optionally contains at least one selectable drug resistance marker for isolating cells that have been modified by the compositions and methods of this disclosure. Selectable drug resistance markers of the present disclosure include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

Those of skill in the art are familiar with the numerous expression systems available for expressing nucleic acid or polynucleotide molecules. Alternatively, a nucleic acid of the disclosure can be expressed in a host cell by turning on (by manipulation) the host cell containing endogenous DNA encoding a nucleic acid or polynucleotide of the disclosure. Such methods are well known in the art, for example U.S. Pat. , which are incorporated herein by reference in their entireties.

Examples of cell cultures useful for the production of nucleic acid and polynucleotide molecules of the disclosure, particular portions or variants thereof, are bacterial, yeast, and mammalian cells known in the art. Mammalian cell lines are often in the form of monolayers of cells, but mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines have been developed in the art, including COS-1 (eg ATCCCRL1650), COS-7 (eg ATCCCRL-1651), HEK293, BHK21 (eg ATCCCRL-10), CHO (eg ATCCCRL1610). , and BSC-1 (eg, ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hepG2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells, etc., which include, for example, Readily available from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2/0-Ag14 cells (ATCC Accession No. CRL-1851). In preferred embodiments, the recombinant cells are P3X63Ab8.653 or SP2/0-Ag14 cells.

Expression vectors for these cells can include, but are not limited to, one or more of the following expression control sequences: promoters such as the late or early SV40 promoter, the CMV promoter (US Pat. 5,168,062; 5,385,839), HSVtk promoter, pgk (phosphoglycerate kinase) promoter, EF-1 alpha promoter (US Pat. No. 5,266,491), at least one human promoter enhancer and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (eg, SV40 large T Ag poly A addition site), and transcription termination sequences. See, eg, Ausubel et al., supra; Sambrook, et al. (supra). Other cells useful for the production of nucleic acids or proteins of the present disclosure are known and/or can be obtained, for example, from the American Type Culture Collection's Catalog of Cell Lines and Hybridomas (www.atcc.org) or other known or commercially available information. available from sources.

When eukaryotic host cells are used, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for correct splicing of transcripts can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, genetic sequences that control replication in the host cell can be incorporated into the vector, as is known in the art.

The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, normally associates with or interacts with a polynucleotide or protein found in its natural environment substantially or essentially free of ingredients; Thus, an isolated or purified polynucleotide or protein, if produced by recombinant techniques, will be substantially free of other cellular material or culture medium and, if chemically synthesized, chemical precursors or other substantially free of chemicals. Optimally, an "isolated" polynucleotide is the sequence that naturally flanks the polynucleotide (i.e., the sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. (optimally, protein coding sequences). For example, in various embodiments, the isolated polynucleotide has about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or It can contain less than 0.1 kb of nucleotide sequence. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the present disclosure or biologically active portion thereof is produced recombinantly, optimally the culture medium contains about 30%, 20%, 10% of chemical precursors or chemicals that are not the protein of interest. %, 5%, or 1% (by dry weight).

The present disclosure provides fragments and variants of the disclosed DNA sequences and the proteins encoded by those DNA sequences. The term "fragment" as used throughout this disclosure refers to a portion of the DNA sequence or a portion of the amino acid sequence, and thus the protein encoded thereby. A fragment of a DNA sequence that includes a coding sequence can encode a protein fragment that retains the biological activity of the native protein and thus can encode DNA recognition or binding activity for the target DNA sequences described herein. Alternatively, fragments of DNA sequences useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to a full-length polynucleotide of the present disclosure.

A nucleic acid or protein of the disclosure can be constructed by a modular approach involving pre-assembly of monomeric and/or repeating units in a target vector, which can then be assembled into the final destination vector. Polypeptides of the disclosure can comprise repeating monomers of the disclosure and can be constructed by a modular approach involving pre-assembly of repeating units in a target vector, which is then assembled into the final destination vector. be able to. The disclosure provides polypeptides produced by this method, as well as nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells containing nucleic acid sequences encoding polypeptides produced by this modular approach.

The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" excludes other elements of essential importance to the combination when used for its intended purpose. shall mean. Thus, a composition consisting essentially of the elements defined herein would not exclude trace contaminants or inert carriers. “Consisting of” shall mean excluding more than trace amounts of other ingredients and substantial method steps. Aspects defined by each of these transition terms are within the scope of this disclosure.

As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. When the polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA in eukaryotic cells.

"Gene expression" refers to the conversion of information contained in a gene into a gene product. A gene product is a direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or produced by translation of mRNA. protein. Gene products include RNA modified by processes such as capping, polyadenylation, methylation, editing, and modified by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, glycosylation, etc. It also contains protein.

"Modulation" or "regulation" of gene expression refers to changes in the activity of a gene. Modulation of expression includes, but is not limited to, gene activation and gene repression.

The term "operatively linked" or its equivalent (e.g., "linked operatively") means that two or more molecules interact to form one or It means positioned relative to each other such that it can affect the function resulting from both molecules or their combination. In some embodiments, a transgene sequence, or any other sequence, is said to be operably linked to a promoter sequence if the promoter sequence controls expression of the transgene sequence, or any other sequence. In some embodiments, a transposase sequence is said to be operably linked to a promoter sequence when the promoter sequence controls the expression of the transposase sequence.

Non-covalent linking moieties and methods of making and using non-covalent linking moieties are disclosed. Various components, as described herein, can take a variety of different forms. For example, non-covalently linked (ie, operably linked) proteins can be used to allow transient interactions that circumvent one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows functional association only or primarily in situations where such association is required for the desired activity. Binding may be of sufficient duration to allow the desired effect.

The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. Depiction of a single strand also defines the sequence of the complementary strand. Nucleic acids can thus also include the depicted single-stranded complementary strand. Nucleic acids of the present disclosure also encompass substantially identical nucleic acids and complements thereof that retain the same structure or encode the same protein.

Nucleic acids of the disclosure can be single-stranded or double-stranded. Nucleic acids of the present disclosure can contain double-stranded sequences even though the molecule is predominantly single-stranded. Nucleic acids of the present disclosure can contain single-stranded sequences even though the majority of the molecule is double-stranded. Nucleic acids of the disclosure can comprise genomic DNA, cDNA, RNA, or hybrids thereof. A nucleic acid of the present disclosure can contain a combination of deoxyribonucleotides and ribonucleotides. Nucleic acids of the present disclosure can contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids of the disclosure can be synthesized to include unnatural amino acid modifications. Nucleic acids of the present disclosure can be obtained by chemical synthetic methods or recombinant methods.

Nucleic acids of the present disclosure may be non-naturally occurring either in their entirety or any portion thereof. Nucleic acids of the present disclosure can contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, which can render the entire nucleic acid sequence non-naturally occurring. A nucleic acid of the present disclosure can contain one or more overlapping, inverted, or repeated sequences, the resulting sequence being non-natural, rendering the entire nucleic acid sequence non-natural. can. Nucleic acids of the present disclosure can comprise non-naturally occurring modified, artificial, or synthetic nucleotides, and can render the entire nucleic acid sequence non-naturally occurring.

Given the redundancy of the genetic code, multiple nucleotide sequences can encode a given protein. All such nucleotide sequences are contemplated herein.

As used throughout this disclosure, the term "operably linked" refers to expression of a gene under control of a spatially linked promoter. Promoters can be positioned 5' (upstream) or 3' (downstream) of the gene under their control. The distance between a promoter and a gene can be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Variations in the distance between promoter and gene can be accommodated without loss of promoter function.

The term "promoter" as used throughout this disclosure refers to synthetic or naturally occurring molecules capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and/or alter its spatial and/or temporal expression. A promoter can also include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. Promoters may be responsive to the cell, tissue, or organ in which expression occurs, or to the developmental stage in which expression occurs, or to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Expression can be regulated constitutively or differentially. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMVIE promoter, EF-1 alpha promoter. , the CAG promoter, the SV40 early promoter, or the SV40 late promoter and the CMVIE promoter.

As used throughout this disclosure, the term "substantially complementary" means that the first sequence is 22,23,24,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,180,270,360,450,540, or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or Refers to 99% identity, or that two sequences hybridize under stringent hybridization conditions.

The term "substantially identical" as used throughout this disclosure means that the first and second sequences are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, at least 60%, 65%, 70% for a nucleic acid over a region of 540 or more nucleotides or amino acids or where the first sequence is substantially complementary to the complement of the second sequence , 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical.

As used throughout this disclosure, the term "variant" when used to describe a nucleic acid includes (i) a portion or fragment of the referenced nucleotide sequence; (ii) the complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) the referenced nucleic acid, its complement, or a sequence substantially identical thereto, under stringent conditions. refers to a nucleic acid that hybridizes with.

The term "vector" as used throughout this disclosure refers to a nucleic acid sequence containing an origin of replication. Vectors may be viral vectors, bacteriophages, bacterial artificial chromosomes or yeast artificial chromosomes. A vector can be a DNA vector or an RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can include a combination of amino acids and DNA sequences, RNA sequences, or both DNA and RNA sequences.

As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but at least one biological A peptide or polypeptide that retains biological activity. A variant can also refer to a protein having an amino acid sequence that is substantially identical to a referenced protein having an amino acid sequence that retains at least one biological activity.

Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, extent and distribution of charged regions) are typically recognized in the art as involving minor changes. recognized. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of amino acids is based on consideration of their hydrophobicity and charge. Amino acids of similar hydropathic index can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows the calculation of the maximum local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. is. See U.S. Pat. No. 4,554,101. which is incorporated herein by reference in its entirety.

Substitution of amino acids having similar hydrophilicity values can result in peptides that retain biological activity, eg, immunogenicity. Substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are those of amino acids, particularly the side chains of those amino acids, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties. It is understood to depend on relative similarity.

As used herein, "conservative" amino acid substitutions may be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and/or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modification of a polynucleotide encoding a polypeptide of the disclosure. Amino acids can be classified according to their physical properties and contribution to the secondary and tertiary structure of proteins. A conservative substitution is the replacement of one amino acid with another amino acid that has similar properties. Examples of conservative substitutions are shown in Table A.

Alternatively, conservative amino acids are grouped as shown in Table B, as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77). can do.

Alternatively, exemplary conservative substitutions are shown in Table C.

Polypeptides of the present disclosure include polypeptides having one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as modifications other than insertions, deletions, or substitutions of amino acid residues. It should be understood that it is intended to include A polypeptide or nucleic acid of the disclosure can contain one or more conservative substitutions.

As used throughout this disclosure, the term "two or more" of the foregoing amino acid substitutions includes Refers to 16, 17, 18, 19, or 20 or more listed amino acid substitutions. The term "two or more" may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

The polypeptides and proteins of this disclosure may not occur in nature, either in their entirety or any portion thereof. The polypeptides and proteins of this disclosure can contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, rendering the entire amino acid sequence non-naturally occurring. The polypeptides and proteins of this disclosure can contain one or more duplicate, inverted, or repeated sequences, the resulting sequence being non-natural, rendering the entire amino acid sequence non-natural. The polypeptides and proteins of this disclosure can include non-naturally occurring modified, artificial, or synthetic amino acids, and the entire amino acid sequence can be non-naturally occurring.

As used throughout this disclosure, "sequence identity" is a stand-alone sequence for blasting two sequences, which can be retrieved from the ftp site of the National Center for Biotechnology Information (NCBI). An executable BLAST engine program (bl2seq) can be used to determine using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). incorporated in the book). The terms "identical" or "identity" when used in the context of two or more nucleic acid or polypeptide sequences refer to a specified percentage of residues that are the same over a specified region of each sequence. The percent is obtained by optimally aligning the two sequences, comparing the two sequences in a designated region, determining the number of positions where identical residues are present in both sequences, and obtaining the number of matched positions. , the number of matched positions divided by the total number of positions within the designated region, and the result multiplied by 100 to obtain the percent sequence identity. If the two sequences differ in length, or if the alignment produces one or more staggered ends and the designated comparison region includes only a single sequence, the residues of a single sequence are in the denominator. Included, but not included in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

The term "endogenous" as used throughout this disclosure refers to nucleic acid or protein sequences that are naturally associated with the target gene or host cell into which it is introduced.

As used throughout this disclosure, the term "exogenous" refers to nucleic acid or protein sequences that are not naturally associated with the target gene or the host cell into which it is introduced, and are non-naturally occurring sequences of naturally occurring nucleic acids, e.g., DNA sequences. Includes naturally occurring nucleic acid sequences located in multiple copies or at non-naturally occurring genomic locations.

The present disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "introducing" is meant presenting a polynucleotide construct to a cell such that the polynucleotide construct can access the interior of the host cell. The methods of the present disclosure do not rely on a particular method for introducing the polynucleotide construct into the host cell, but only on the polynucleotide construct reaching the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and viral methods. Including mediation methods.

As used herein, the term "subject" is interchangeable with the term "subject in need thereof," both of which refer to a subject who has a disease or is at high risk for developing a disease. . "Subject" includes mammals. The mammal may be, for example, a human or a suitable non-human mammal such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. The subject may be a bird or poultry. In one embodiment, the mammal is human.

As used herein, the terms "treating" or "treating" describe the management and care of a patient aimed at combating a disease, condition, or disorder; administration of a compound of the disclosure, or administration of a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate symptoms or complications of or to eliminate a disease, condition, or disorder including. The term "treating" can also include in vitro cell or animal model treatment.

Example 1 - In Vivo Expression of Transgenes Mediated by Viral Vectors of the Disclosure

In the following non-limiting example, mice were treated with the viral vectors of the present disclosure and the expression of transgenes contained in the viral vectors was tracked.

Neonatal mice were divided into four different treatment groups.

Mice in treatment group #1 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 1 at a dose of 3.3×10 ¹³ vector genomes (vg)/kg.

Mice in treatment group #2 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 8 at a dose of 3.3×10 ¹³ vg/kg.

Mice in treatment group #3 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 1.1×10 ¹³ vg/kg. The AAV transposase polynucleotide contained transposase sequences encoding SPB transposase.

Mice in treatment group #4 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 1.1×10 ¹³ vg/kg. AAV Transposase Polynucleotides Contained Transposase Sequences Encoding SPB Transposase

Bioluminescence (BLI) signals were then measured in mice for 35 days after viral vector administration to measure expression of the transgene encoded in the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in FIG. In FIG. 10, treatment group #1 is called HLP-OTC, treatment group #2 is called TBG-OTC, treatment group #3 is called HLP-OTC+SPB, and treatment group #4 is called TBG-OTC+SPB.

As shown in Figure 10, mice in treatment groups #3 and #4 show an increase in BLI levels over the 35 day period. Without wishing to be bound by theory, these results suggest that co-administration of an AAVpiggyBac transposon vector and an AAV transposase vector results in integration of the transgene of the AAVpiggyBac transposon vector into the genome of the host and expression of the transgene. results in an increase and persistence of In addition, increased expression of the transgene was observed in treatment group #4 that received the AAVpiggyBac transposon vector containing the TBG promoter. Without wishing to be bound by theory, these results indicate that use of the TBG promoter can provide increased transgene expression that occurs shortly after administration. Such activity is particularly advantageous in clinical settings where early onset patients are being treated.

Example 2 - In Vivo Expression of Transgenes Mediated by Viral Vectors of the Disclosure at Different Concentrations

In the following non-limiting examples, mice were treated with various concentrations of viral vectors of the present disclosure and the expression of transgenes contained in the viral vectors was tracked.

Mice in this study received either:
a) increasing concentrations of the AAVpiggyBac transposon vector alone comprising the AAVpiggyBac transposon polynucleotides described in FIG. 8, or b) the AAVpiggyBac transposon polynucleotides described in FIG. AAV piggyBac transposon vectors containing increasing concentrations, where the AAV transposase polynucleotide contained the transposase sequence encoding the SPB transposase.

Twenty-one days after viral vector administration, BLI was measured in mice to measure the expression of the transgene encoded in the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in FIG. As shown in Figure 11, higher levels of transgene expression were achieved at lower doses when both the AAVpiggyBac transposon vector and the AAV transposase vector were co-administered. Without wishing to be bound by theory, these results suggest that co-administration of an AAVpiggyBac transposon vector and an AAV transposase vector results in integration of the transgene of the AAVpiggyBac transposon vector into the genome of the host and expression of the transgene. results in an increase and persistence of This is particularly advantageous in a clinical setting as it can reduce the total dose of AAV that needs to be administered to a subject and helps avoid the negative side effects normally associated with administration of AAV vectors.

Example 3 - Treatment of Otc ^spf-ash mice with viral vectors of the disclosure

In the following non-limiting example, Otc ^spf-ash mice were treated with viral vectors of the disclosure.

As will be appreciated by those skilled in the art, the Otc ^spf-ash mouse is a widely used model of urea cycle disorders, including OTC deficiency and chronic hyperammonemia. The mouse contains a mutation (c.386G>A; p.R129H) in the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice junction and creating a 48 bp cryptic splice junction into the adjacent intron. result in the partial use of

Newborn Otc ^spf-ash mice were divided into two different treatment groups.

Mice in treatment group #1 were administered an AAVpiggyBac transposon vector containing the AAVpiggyBac ^transposon polynucleotide described in FIG. AAV transposase vectors containing transposase polynucleotides were administered at a dose of 3.3×10 ¹³ vg/kg. Therefore, treatment group #1 mice were treated with AAVpiggyBac transposon vector and AAV transposase vector in a 1:1 dose ratio for a total AAV dose of 6.6×10 ¹³ vg/kg. The AAV transposase polynucleotide contained transgene sequences encoding human OTC, allowing it to be distinguished from endogenous mouse OTC.

Mice in treatment group #2 were administered an AAVpiggyBac transposon vector containing the AAVpiggyBac ^transposon polynucleotide described in FIG. AAV transposase vectors containing transposase polynucleotides were administered at a dose of 1.7×10 ¹³ vg/kg. Therefore, treatment group #2 mice were treated with AAVpiggyBac transposon vector and AAV transposase vector in a 1:1 dose ratio for a total AAV dose of 5×10 ¹³ vg/kg. The AAV transposase polynucleotide contained transgene sequences encoding human OTC, allowing it to be distinguished from endogenous mouse OTC.

After administration of the viral vector, BLI was measured in mice to measure the expression of the transgene encoded in the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in FIG. As shown in FIG. High levels of transgene expression were measured in both treatment groups.

After viral vector administration, the amount of non-integrating vector copy number per diploid genome was determined for both the AAV piggyBAC transposon and AAV transposase vectors. The results of this analysis are shown in FIG. As shown in Figure 13, the amount of unintegrated AAV transposase vector decreased with aging of the mice. In addition, the amount of non-integrated AAV transposase vector was low compared to the amount of non-integrated AAVpiggyBac transposon vector even at day 7.

After viral vector administration, the amount of non-integrated AAVpiggyBac transposon vector copy number per diploid genome and the amount of integrated AAVpiggyBac transposon vector copy number was determined. The results of this analysis are shown in FIG. As shown in Figure 14, integrated AAV piggbyBac transposon vector was detected 21 days after treatment. In addition, incorporation was more consistent in treatment group #1 (2:1 OTC:SPB) compared to treatment group #2 (2:2 OTC:SPB). While not wishing to be bound by theory, the results of Figure 14, particularly transposon vector integration, demonstrate successful transposition of the transposon in vivo.

The number of integrated sites was also assayed by LM-PCR on days 21 and 43. Briefly, 2 μg of genomic DNA was isolated from mouse liver tissue and randomly sheared by sonication. The resulting ends were ligated with a unique molecular identifier (UMI). Two rounds of PCR amplification were performed. Final PCR products were subjected to Illumina paired-end sequencing. The site of integration was determined by checking two breakpoints. The results of this PCR analysis are shown in Tables 1 and 2. Without wishing to be bound by theory, the results presented in Tables 1 and 2 demonstrate successful transposition and integration of the transposon in vivo.

After administration of the viral vector, the amount of human OTC mRNA and SPB mRNA relative to the level of mouse OTC mRNA was measured in mice. The results of this analysis are shown in FIG. As shown in Figure 15, viral vector-treated mice express high amounts of human OTC mRNA. In addition, SPB mRNA levels decrease with age. Without wishing to be bound by theory, this reduction of SPB mRNA may be advantageous in the clinical setting to avoid off-target translocation effects after initial treatment. A correlation analysis between human OTC and SPB mRNA and total vector copy number per diploid genome was also performed. The results of this analysis are shown in FIG. As shown in Figure 16, human OTC and SPB mRNA levels correlated with the corresponding vector copy numbers.

Twenty-one days after treatment, liver samples were taken from mice and analyzed for GFP expression. Hepatocytes in samples taken from both treatment group #1 and treatment group #2 showed strong GFP expression.

Example 4 - Treatment of Inducible Hyperammonemia Mouse Model with Viral Vectors of the Disclosure

In the following non-limiting example, Otc ^spf-ash mice were treated with viral vectors of the disclosure and shRNA was used to create an induced hyperammonemia disease model.

Newborn Otc ^spf-ash mice were divided into two different treatment groups.

Mice in treatment group #1 received an AAVpiggyBac transposon vector containing the AAVpiggyBac ^transposon polynucleotide described in FIG. AAV transposase vectors containing polynucleotides were administered at a dose of 3.3×10 ¹³ vg/kg. Therefore, treatment group #1 mice were treated with AAVpiggyBac transposon vector and AAV transposase vector in a 1:1 dose ratio for a total AAV dose of 6.6×10 ¹³ vg/kg. The AAV transposase polynucleotide contained transgene sequences encoding human OTC, allowing it to be distinguished from endogenous mouse OTC.

Mice in treatment group #2 were administered an AAVpiggyBac transposon vector containing the AAVpiggyBac ^transposon polynucleotide described in FIG. AAV transposase vectors containing transposase polynucleotides were administered at a dose of 1.7×10 ¹³ vg/kg. Therefore, treatment group #2 mice were treated with AAVpiggyBac transposon vector and AAV transposase vector in a 2:1 dose ratio for a total AAV dose of 5×10 ¹³ vg/kg. The AAV transposase polynucleotide contained transgene sequences encoding human OTC, allowing it to be distinguished from endogenous mouse OTC.

At 38 days post-treatment, subsets in each treatment group were either:
a) further rest b) administration of a dose of shRNA targeting mouse OTC.

As a control and for comparison, similarly aged Otc ^spf-ash mice that were not treated with viral vector were also administered doses of shRNA targeting mouse OTC.

FIG. 17 shows mice in treatment group #1 either left untreated (2:2 OTC:SPB) or additionally administered a dose of shRNA targeting mouse OTC (2:2 OTC:SPB+shRNA). shows the survival probability of FIG. 17 also shows the survival probabilities of similarly aged Otc ^spf-ash mice that were not treated with viral vectors and received doses of shRNAs targeting mouse OTC. FIG. 18 shows plasma ammonia concentrations in the aforementioned mouse groups. As shown in Figures 17 and 18, the detrimental effects of shRNA administration are delayed in mice treated with viral vectors.

Example 5 - In Vivo Expression of Transgenes Operably Linked to Different Promoter Sequences in AAVpiggyBac Transposon Vectors of the Disclosure

In the following non-limiting examples, mice were treated with viral vectors of the present disclosure containing transgenes operably linked to either the HLP promoter, LP1 promoter, or TBG promoter sequences. Expression of transgenes contained in viral vectors was followed to determine the efficiency with which each promoter was able to drive transgene expression in vivo, particularly in the liver.

Neonatal wild-type and adult wild-type and neonatal Otc ^spf-ash mice were divided into 12 different treatment groups.

Treatment groups #1-#6 contained newborn wild-type mice.

Mice in treatment group #1 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 1 at a dose of 5×10 ¹³ vg/kg.

Mice in treatment group #2 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 1.7×10 ¹³ vg/kg. The AAV transposase polynucleotide contained a transposase sequence encoding SPB transposase operably linked to the HLP promoter.

Mice in treatment group #3 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 8 at a dose of 5×10 ¹³ vg/kg.

Mice in treatment group #4 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 1.7×10 ¹³ vg/kg. The AAV transposase polynucleotide contained a transposase sequence encoding SPB transposase operably linked to the HLP promoter.

Mice in treatment group #5 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 9 at a dose of 95×10 ¹³ vg/kg.

Mice in treatment group #6 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 1.7×10 ¹³ vg/kg. The AAV transposase polynucleotide contained a transposase sequence encoding SPB transposase operably linked to the HLP promoter.

Liver bioluminescence (BLI) signals were then measured in mice in treatment groups #1-#6 for 42 days after viral vector administration to measure expression of the transgene encoded in the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in FIG. In FIG. 19, treatment group #1 is called HLP-OTC, treatment group #2 is called HLP-OTC+SPB, treatment group #3 is called TBG-OTC, treatment group #4 is called TBG-OTC+SPB, and treatment group #5 is called LP1-OTC+SPB and treatment group #6 is called LP1-OTC+SPB. As shown in Figure 19, the TBG promoter most efficiently drove transgene expression, followed by the LP1 promoter and then the HLP promoter. Increased and sustained expression was observed in treatment groups #2, #4, and #6. Without wishing to be bound by theory, these results suggest that co-administration of an AAVpiggyBac transposon vector and an AAV transposase vector results in integration of the transgene of the AAVpiggyBac transposon vector into the genome of the host and expression of the transgene. results in an increase and persistence of Integration of the transposon vector observed in Figure 19 indicates successful transposition of the transposon in vivo.

The amounts of human OTC mRNA and SPB mRNA relative to the levels of mouse OTC mRNA were also measured in mice in treatment groups #1-#6. The results of this analysis are shown in Figure 20 (human OTC mRNA) and Figure 21 (SPB mRNA). Similar to the results shown in Figure 19, the results in Figures 20 and 21 show that the TBG promoter yields the highest levels of transgene mRNA, followed by the LP1 promoter and then the HLP promoter.

Twenty-one days after viral vector administration, the amount of human OTC protein relative to the amount of mouse OTC protein was also measured. The results of this analysis are shown in FIG. Similar to the results shown in Figures 19-21, the results shown in Figure 22 indicate that the TBG promoter yields the highest levels of human OTC protein, followed by the LP1 promoter and then the HLP promoter.

In addition, hepatocytes from treatment group #1 and treatment #2 mice were also analyzed by immunohistochemistry and stained for GFP. Briefly, liver tissue was harvested on day 21, fixed in 10% neutral buffered formalin, embedded in paraffin, and stained. Immunohistochemistry results are shown in Figure 27, indicating that higher levels of GFP were observed in treatment group #2. Without wishing to be bound by theory, these results suggest that co-administration of an AAVpiggyBac transposon vector and an AAV transposase vector results in integration of the transgene of the AAVpiggyBac transposon vector into the genome of the host and expression of the transgene. results in an increase and persistence of

Additionally, the number of integrated sites in the genome of treated mice was assayed by LM-PCR. Briefly, 2 μg of genomic DNA was isolated from mouse liver tissue and randomly sheared by sonication. A unique molecular identifier (UMI) was ligated to the resulting ends. Two rounds of PCR amplification were performed. Final PCR products were subjected to Illumina paired-end sequencing. The total number of unique integration sites was determined by unilateral breakpoints with 2 or more UMIs. The results of this analysis are shown in Table 3.

Treatment groups #7-#12 contained adult wild-type mice.

Mice in treatment group #7 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 1 at a dose of 2×10 ¹³ vg/kg.

Mice in treatment group #8 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 0.7×10 ¹³ vg/kg. The AAV transposase polynucleotide contained a transposase sequence encoding SPB transposase operably linked to the HLP promoter.

Mice in treatment group #9 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 8 at a dose of 2×10 ¹³ vg/kg.

Mice in treatment group #10 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 0.7×10 ¹³ vg/kg. The AAV transposase polynucleotide contained a transposase sequence encoding SPB transposase operably linked to the HLP promoter.

Mice in treatment group #11 received an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide described in FIG. 9 at a dose of 2×10 ¹³ vg/kg.

Mice in treatment group #12 received the AAVpiggyBac transposon vector containing the AAVpiggyBac transposon ^{polynucleotide} described in FIG. Vector was administered at a dose of 0.7×10 ¹³ vg/kg. The AAV transposase polynucleotide contained a transposase sequence encoding SPB transposase operably linked to the HLP promoter.

Liver bioluminescence (BLI) signals were then measured in mice in treatment groups #7-#12 on days 7 and 14 after viral vector administration to determine the presence of the transgene encoded in the AAVpiggyBac transposon polynucleotide. Expression was measured. The results of this analysis are shown in FIG. In FIG. 23, treatment group #7 is called HLP-OTC, treatment group #8 is called HLP-OTC+SPB, treatment group #9 is called TBG-OTC, treatment group #10 is called TBG-OTC+SPB, and treatment group #11 is called LP1-OTC+SPB and treatment group #12 is called LP1-OTC+SPB. As shown in Figure 23, similar strengths of transgene expression were observed for each of the promoters.

The amount of human OTC mRNA and SPB mRNA relative to the level of mouse OTC mRNA was also measured in mice in treatment groups #7-#12 14 days after viral vector administration. The results of this analysis are shown in Figure 24 (human OTC mRNA) and Figure 25 (SPB mRNA). As shown in Figures 24 and 25, similar levels of human OTC were observed for the HLP and LP1 promoters, with the strongest T expression observed for the BG promoter.

Fourteen days after viral vector administration, the amount of human OTC protein relative to the amount of mouse OTC protein was also measured. The results of this analysis are shown in 26. Similar to the results shown in Figures 19-21, the results shown in Figure 22 indicate that the TBG promoter yields the highest levels of human OTC protein, followed by the LP1 promoter and then the HLP promoter.

Equivalents The foregoing description has been presented for purposes of illustration only and is not intended to limit the disclosure to the precise forms disclosed. The details of one or more implementations of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described herein. Other features, objects, and advantages of the disclosure will be apparent from the description and claims. In this specification and the appended claims, singular forms include plural forms unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference.

Claims (24)

An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 126;
e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96;
i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:129; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4. 2. The AAVpiggyBac transposon polynucleotide of claim 1, comprising the nucleic acid sequence of SEQ ID NO:138. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 132;
e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96;
i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:130; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4. 4. The AAVpiggyBac transposon polynucleotide of claim 3, comprising the nucleic acid sequence of SEQ ID NO:139. An AAV piggyBac transposon polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:3;
b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 125;
c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:7;
d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 13;
e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO:22;
f) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO:97;
g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:8;
h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO:96;
i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:131; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4. 6. The AAVpiggyBac transposon polynucleotide of claim 5, comprising the nucleic acid sequence of SEQ ID NO:140. A vector comprising an AAVpiggyBac transposon polynucleotide according to any preceding claim. 8. The vector of claim 7, wherein said vector is a viral vector, preferably said viral vector is an AAV viral vector. 9. The vector of claim 8, wherein the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector. Vector according to claim 8, wherein said AAV viral vector is an AAV-KP-1 or AAV-NP59 viral vector, preferably said AAV viral vector is an AAV-KP-1 viral vector. A composition comprising the vector according to any one of claims 7-10. An AAV transposase polynucleotide comprising, in the 5' to 3' direction:
a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 127;
b) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 126;
c) at least one transposase sequence comprising the nucleic acid sequence of SEQ ID NO:48;
d) a poly A sequence comprising the nucleic acid sequence of SEQ ID NO: 136;
e) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO:137; and f) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO:4. 13. The AAV transposase polynucleotide of claim 12, comprising the nucleic acid sequence of SEQ ID NO:144. 14. A vector comprising the AAV transposase polynucleotide of claim 12 or claim 13. 15. The vector of claim 14, wherein said vector is a viral vector, preferably said viral vector is an AAV viral vector. 16. The vector of claim 15, wherein the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector. 16. The vector of claim 15, wherein said AAV viral vector is an AAV-KP-1 or AAV-NP59 viral vector, preferably said AAV viral vector is an AAV-KP-1 viral vector. A composition comprising the vector of any one of claims 14-17. at least one metabolic activity in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of at least one of the polynucleotides, vectors, or compositions of any one of the preceding claims. A method of treating liver damage (MLD). A method of treating at least one MLD in a subject in need thereof comprising administering to the subject:
a) a polynucleotide according to any one of claims 1 to 6, a vector according to any one of claims 7 to 10, or a composition according to claim 11, and b) claims 12 to A polynucleotide according to any one of claims 13, a vector according to any one of claims 14-17, or a composition according to claim 18. 12. Use of a polynucleotide, vector or composition according to any one of the preceding claims for the treatment of at least one MLD in a subject in need thereof, said polynucleotide, vector or Use wherein the composition is for administering at least one therapeutically effective amount thereof to a subject. for use in treating at least one MLD in a subject in need thereof;
a) a polynucleotide according to any one of claims 1 to 6, a vector according to any one of claims 7 to 10, or a composition according to claim 11;
b) in combination with a polynucleotide according to any one of claims 12-13, a vector according to any one of claims 14-17 or a composition according to claim 18. at least one MLD, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) ) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome) , methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis 1 (PFIC3), or any combination thereof. 24. A method or use according to claim 23, wherein said MLD is OTC deficiency.

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